Synthesis of promising brominated flavonoids as antidiabetic and anti-glycation agents

Synthesis of promising brominated flavonoids as antidiabetic and anti-glycation agents | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Synthesis of promising brominated flavonoids as antidiabetic and anti-glycation agents Rita Hairani, Warinthorn Chavasiri This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6452882/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 15 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted 11 You are reading this latest preprint version Abstract Twelve selected natural flavonoids were investigated for their antidiabetic effects on α -glucosidase and α -amylase, as well as anti-glycation activity. A series of brominated analogues from these selected flavonoids were synthesized. Two new semisynthetic compounds including 6,8-dibromoluteolin ( F3a ) and 6,8-dibromoalpinetin ( F10a ) have been synthesized. 8-Bromobaicalein ( F4a , IC 50 = 0.52 ± 0.05 µM) and 6,8-dibromoluteolin ( F3a , IC 50 = 0.99 ± 0.12 µM) were found as mixed-type potent agents on α -glucosidase and α -amylase inhibitory activities, respectively. In addition, 6,8-dibromochrysin ( F1a , IC 50 = 50.90 ± 0.98 µM) was found to be the most potent compound for the inhibition of Bovine Serum Albumin - glycation (BSA-glycation) mediated methylglyoxal. Biological sciences/Drug discovery Health sciences/Diseases Physical sciences/Chemistry 8-bromobaicalein 6 8-dibromoluteolin 6 8-dibromochrysin α-glucosidase α-amylase anti-glycation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Diabetes mellitus (DM), a chronic metabolic disorder with a high global prevalence, is divided into two types namely type 1 DM which is generated by dysfunction of β -pancreatic cells, and type 2 DM which is caused by impairment of insulin secretion due to insulin resistance and results in disturbances in glucose homeostasis. 1 , 2 Type 2 DM is recognized as a major threat to human health and development and has emerged as one of the most prevalent public health issues. 3 – 5 In the case of type 2 DM, the condition could lead to an increase in blood glucose levels. The inhibition of the activities of α -glucosidase and α -amylase enzymes, which catalyze carbohydrate hydrolysis, is believed as one approach for controlling blood glucose levels. 6 Over the last two decades, the demand for antidiabetic medications has risen quickly as DM becomes more prevalent. 7 , 8 One of the drugs used to treat type 2 DM and known to inhibit α -glucosidase and α -amylase activities, i.e. acarbose has some drawbacks including the side effects related to diarrhea and flatulence. 9 Moreover, its production still faces significant challenges that drive up manufacturing costs, one of which is through genetic engineering. 10 On the other hand, diabetic patients tend to develop vascular complications, which are a leading cause of morbidity and mortality. 11 Chronic hyperglycemia resulting in advanced glycation end-products (AGEs) facilitates diabetes-related complications, such as diabetic retinopathy, nephropathy, peripheral neuropathy, atherosclerosis, and other complications. 12 , 13 The primary serum protein, albumin, has a number of roles and is very susceptible to several environmental factors, with glucose being one of the most significant. 14 During long-standing hyperglycemia states in DM, the steadily increasing glucose level tends to form covalent adducts with plasma proteins via a non-enzymatic process named glycation or Maillard reaction, resulting in the formation of AGEs. 15 A class of compounds has been identified either to prevent the formation or to degrade existing AGEs, which have been manufactured and patented. 16 , 17 Aminoguanidine, known as the first AGEs inhibitor, could trap or scavenge the reactive carbonyl intermediates such as glyoxal and methylglyoxal (MG) in the glycation process. 18 However, a previous study revealed that the use of a high concentration of aminoguanidine was required to trap the reactive species due to its half-life in plasma is short. 19 This is one of the disadvantages of aminoguanidine which could generate serious toxicity when administered for diabetic nephropathy. In terms of discovering the effective drugs that could overcome the side effects of the reference drugs mentioned above, the researchers have a broad interest in studying the biological activities of natural products since they are well-known to have withdrawal symptoms. 20 , 21 Therefore, it is noteworthy to discover and further investigate the potency of secondary metabolites from natural products as new promising antidiabetic and anti-glycation agents. Flavonoids, one of the secondary metabolites, are known to possess antidiabetic and anti-glycation properties. 22 , 23 Due to their abundance in nature with known biological activities, flavones, flavonols, and flavanones can be highlighted among the several subclasses of flavonoids. Hence, it is exciting to explore the effectiveness of these constituents on antidiabetic and anti-glycation activities. Whereas, in this study, twelve selected natural flavonoids as portrayed in Fig. 1 were examined. Moreover, halogenated secondary metabolites have received increased attention in drug discovery and medicinal chemistry in recent decades. The insertion of halogen atoms into the moiety structures of natural products or synthetic compounds have known to affect biological activities. 24 Halogens, especially lighter fluorine and chlorine, are widely used substituents in medicinal chemistry. 25 In the case of bromine, many studies have been reported on the preparation of brominated flavonoids. In addition, a previous study reported that the brominated flavones exhibited better α -glucosidase inhibition compared with their parent compounds and found that the number of bromine atoms on the B-ring affected the biological activity. 26 Hence, this study aims to investigate the effects of bromine atoms in the A-ring of the flavonoid scaffold on α -glucosidase and α -amylase inhibitory activities. Further investigation was carried out by performing the anti-glycation assay to examine whether those brominated flavonoids were also acting as glycation inhibitors which are further useful to treat diabetic complications. To date, this is the first report about the effects of those semisynthetic brominated flavonoids (flavones, flavonols, and flavanones) as antidiabetic and anti-glycation agents. All the selected natural flavonoids and their brominated compounds were examined on inhibitory activities against α -glucosidase from Saccharomyces cerevisiae and α -amylase from porcine pancreas. Moreover, since bovine serum albumin (BSA) could act as a model protein in the in vitro experiment, hence the study also focused on the activity of those compounds toward BSA-glycation mediated MG. Results and Discussion Chemistry In this study, four flavonoids including kaempferol ( F5 ), pinocembrin ( F8 ), pinostrobin ( F9 ), and alpinetin ( F10 ) were isolated from two plants material. The isolation of F5 was carried out by further purification of the MeOH extract of the flowers of Impatiens balsamina L. using a silica gel column to yield F5 as a yellow powder ( 69%). On another hand, three flavanones including F8 , F9 , and F10 were isolated from the MeOH extract of the rhizome of Boesenbergia rotunda (L.) Mansf. The purification of selected fractions by silica gel column and recrystallization furnished a colorless crystal of F8 (46%), pale-yellow powder of F9 (16%), and F10 (8.1%). The structures of these isolated flavonoids were identified by NMR analysis and compared with previously reported data. 27 , 28 Several protocols to introduce the bromine atom on the A-ring of the flavonoid scaffold were carried out. The synthesis of bromoflavonoids including bromoflavones, bromoflavonols, and bromoflavanones are depicted in Fig. 2 . To synthesize bromo derivatives of flavones ( F1a – F3a ), a modified method described by Hairani and Chavasiri was carried out. 29 The reaction was performed in the presence NaBr and oxone in acetone-H 2 O. The desired products F1a , F2a , and F3a were characterized by the absence of signals of 6- and 8- protons. The NMR spectral data of F1a , F2a , and F4a were also confirmed with those of the previous reports. 29 – 31 From this series, F3a is identified as a new semisynthetic compound. The selective bromination of baicalein at the C-8 position was carried out using a modified method described by Lee and coworkers using NBS in the presence of a catalytic amount of concentrated H 2 SO 4 at room temperature to yield F4a and compared its NMR spectra with a previously reported data. 31 While, the preparation of F5a and F6a was performed by using NaBr and oxone in acetone-H 2 O, while, F7a could be attained by using Br 2 in CH 3 COOH. 32 For bromoflavanones ( F8a – F12a ) could also be obtained by using NaBr and oxone in acetone-H 2 O at room temperature for overnight. Among these series, F10a was identified as a new semisynthetic compound. Biological activity of natural flavonoids and their brominated compounds In vitro α-glucosidase inhibitory All selected flavonoids and their bromo derivatives were screened in vitro for inhibitory α -glucosidase activity which performed according to the previously described protocol. 29 Acarbose was used as a positive control. The inhibition results at 50 µ M and IC 50 values for the compounds that exhibited ≥ 50% inhibition toward α -glucosidase is described in Table 1 . The criteria of IC 50 value is categorized as follow: IC 50 200 µ M (not active). Table 1 shows various effects of bromine atoms on the inhibition of α -glucosidase. The introduction of bulky groups such as bromine could be antagonistic or agonistic in comparison with the parent compound. 24 In this screening study, the brominated compounds showed better activities compared with their parent ones. Nonetheless, some derivatives displayed no effects of bromine atoms or relatively have the same activity compared with the starting-material. Table 1 Enzymatic inhibitory activities of natural flavonoids and their brominated compounds. Compound α -Glucosidase α -Amylase % Inhibition at 50 µ M* IC 50 ( µ M)* % Inhibition at 50 µ M* IC 50 ( µ M)* Flavones : Chrysin ( F1 ) 31.21 ± 2.81 n.d. 22.59 ± 0.37 n.d. 6,8-Dibromochrysin ( F1a ) 89.70 ± 1.51 2.97 ± 0.03 11.68 ± 3.47 n.d. Apigenin ( F2 ) 78.64 ± 3.46 22.74 ± 0.08 87.47 ± 0.81 13.18 ± 0.81 6,8-Dibromoapigenin ( F2a ) 81.98 ± 0.84 8.91 ± 0.27 69.28 ± 1.58 n.d. Luteolin ( F3 ) 34.37 ± 2.60 n.d. 97.61 ± 0.13 2.18 ± 0.07 6,8-Dibromoluteolin ( F3a ) 95.08 ± 2.84 15.32 ± 0.38 98.57 ± 1.80 0.99 ± 0.12 Baicalein ( F4 ) 99.42 ± 0.38 0.58 ± 0.01 64.04 ± 0.41 n.d. 8-Bromobaicalein ( F4a ) 99.74 ± 0.11 0.52 ± 0.05 0.00 ± 0.00 n.d. Flavonols : Kaempferol ( F5 ) 39.77 ± 2.76 n.d. 87.30 ± 2.31 10.43 ± 0.49 6,8-Dibromokaempferol ( F5a ) 78.99 ± 0.36 13.89 ± 0.50 98.19 ± 2.82 3.71 ± 0.26 Morin ( F6 ) 97.47 ± 1.40 9.15 ± 0.26 51.82 ± 4.65 n.d. 6,8-Dibromomorin ( F6a ) 99.38 ± 0.77 8.28 ± 0.03 98.65 ± 1.72 8.38 ± 0.69 Quercetin ( F7 ) 98.68 ± 0.03 1.11 ± 0.05 94.06 ± 2.61 11.16 ± 0.32 6,8-Dibromoquercetin ( F7a ) 95.76 ± 1.40 3.90 ± 0.34 71.51 ± 1.86 19.88 ± 0.19 Flavanones : Pinocembrin ( F8 ) 7.14 ± 0.21 n.d. 25.23 ± 1.15 n.d. 6,8-Dibromopinocembrin ( F8a ) 25.84 ± 0.42 n.d. 51.13 ± 1.02 n.d. Pinostrobin ( F9 ) 15.13 ± 3.35 n.d. 12.85 ± 0.16 n.d. 6,8-Dibromopinostrobin ( F9a ) 29.55 ± 0.43 n.d. 20.82 ± 4.55 n.d. Alpinetin ( F10 ) 0.00 ± 0.00 n.d. 34.13 ± 0.98 n.d. 6,8-Dibromoalpinetin ( F10a ) 0.00 ± 0.00 n.d. 43.82 ± 1.69 n.d. Naringenin ( F11 ) 26.53 ± 0.33 n.d. 33.29 ± 0.73 n.d. 6,8-Dibromonaringenin ( F11a ) 80.55 ± 0.29 13.63 ± 0.64 49.46 ± 4.00 n.d. Hesperetin ( F12 ) 45.39 ± 0.52 n.d. 29.90 ± 0.63 n.d. 6,8-Dibromohesperetin ( F12a ) 66.26 ± 2.51 16.70 ± 0.21 40.52 ± 0.66 n.d. Acarbose 37.87 ± 0.13 83.67 ± 0.04 87.92 ± 0.25 6.73 ± 0.54 *Mean ± Standard deviation from two independent experiments and each performed in triplicate, n.d. = not determined For flavones subclasses, baicalein ( F4 ) which consists of three -OH groups on the A-ring exhibited very strong inhibition among flavones with a % inhibition of 99.42 and IC 50 value of 0.58 µ M. This result indicated that the number of the -OH groups on the A-ring of flavones might play a crucial role in the enzyme inhibition due to the fact that these could create more interactions with the enzyme via hydrogen bonds and or electrostatic interactions. 33 Even though apigenin ( F2 ) also has three -OH groups, due to the position of one hydroxyl group on the B-ring, the inhibition of F2 was not as strong as baicalein ( F4 ). It showed relatively strong inhibition with a % inhibition of 78.64 and an IC 50 value of 22.74 µ M. This indicated that the -OH groups of flavones on the A-ring are more favorable for the interaction with the enzyme, whereas 5,6,7-trihydroxyflavone could be crucial substituents for enhancing α -glucosidase inhibitory activity as stated by a previous study. 34 For luteolin ( F3 ) containing four -OH groups exhibited only 34.37% inhibition. This could be concluded that the presence of the -OH group in the 3′-position of the B-ring slightly withdraws the activity of a compound. Comparing the natural flavones and their bromo derivatives, the results showed that the introduction of bromine atoms at positions 6- and 8- on chrysin ( F1a ), apigenin ( F2a ), and luteolin ( F3a ) exhibited better activities compared with their parent compounds chrysin ( F1 ), apigenin ( F2 ), and luteolin ( F3 ) with % inhibition: 89.70, 81.98 and 95.08%, and IC 50 values: 2.97, 15.32 µ M, respectively. In contrast, the brominated baicalein ( F4a ) showed a slight increase or no effect compared with its starting material. This could be indicated that the introduction of bromine atom at position 8- on flavones only give little effect or might not give an advantageous effect on α -glucosidase inhibitory activity. For flavonols, the results showed that the natural flavonols including morin ( F6 ) and quercetin ( F7 ) containing five -OH groups displayed better activities than kaempferol ( F5 ) which only four -OH groups. Even though morin ( F6 ) and quercetin ( F7 ) have the same number of -OH groups, the different positions of the -OH group lead to the different power of compounds to reduce α -glucosidase activity, whereas quercetin ( F7 ) showed strong inhibition with an IC 50 value 1.33 µ M. This implied that the catechol moiety ( ortho -OH groups) in F7 gave the benefit towards the α -glucosidase inhibitory activity over the meta -OH groups. Moreover, for the brominated flavonols, F5a and F6a displayed relatively strong inhibition and were better than their parent compounds with IC 50 values: 13.89 and 8.28 µ M, respectively. In contrast, brominated quercetin ( F7a ) did not give a crucial effect on α -glucosidase activity whereas the activity slightly declines. This behavior might be due to the bulky compounds compared with the starting material which is then difficult to reach the binding site of the enzyme. However, F7a (IC 50 value 3.90 µ M) showed a better effect compared with F5a and F6a . In some cases, the hydrophobicity of the compound played an important effect on enzyme activity which led to an increase in the hydrophobic interaction between the compound and enzyme. The value of log P (octanol-water partition coefficient) could be used to describe how hydrophilic or hydrophobic a compound. Comparing the c Log P values of F6a (1.331) and F7a ( 1.701) whereas F7a was more hydrophobic than F6a , therefore F7a was stronger than F6a due to the possibility of F7a having more hydrophobic interactions with the enzyme. Most of the natural flavanones and their brominated compounds showed weak inhibition, except for compounds F11a and F12a with relatively strong inhibition. This phenomenon indicated that the absence of a double bond at C-2 and C-3 positions led to the lessening of the α -glucosidase inhibitory activity. Compound F11a (IC 50 13.63 µ M) showed better inhibition compared with F12a (IC 50 16.70 µ M) due to the -OH group at 4′-position being replaced by a -OMe group leading to a slight decrease the activity. According to the findings, the structure-activity relationship of natural and brominated flavonoids could be rationalized into several important points as illustrated in Fig. 3 . Furthermore, Lineweaver-Burk plot was established to illustrate the kinetic inhibition of α -glucosidase in the presence of F4a . The plot of 1/v vs 1/[p-NPG] for F4a , gave straight lines that intersected at the same point in the second quadrant, indicating that F4a was a mixed-type inhibitor (Fig. 4 ). This result indicated that F4a could bind with free enzymes, and enzyme-substrate complex to reduce the catalytic activity of α -glucosidase. In addition, the equilibrium constants of binding of F4a to free enzyme ( K i ) and enzyme-substrate complex ( K is ) through plots of slope ( K m /V m ) and vertical intercept (1 /V m ) vs F4a with various concentrations were also determined. From the calculation, the K i value was 1.37 µ M while K is value was 2.53 µ M. The K is value of F4a was higher than its K i value, suggesting that the affinity of F4a with free enzyme was higher than that with the enzyme-substrate complex. In vitro α-amylase inhibitory activity The α -amylase assay was performed in 96-well microplates using a final volume of 200 µ L as the previous method with some modification. 35 , 36 The screening at 50 µ M (Table 1 ) showed that flavone and flavonol sub-classes demonstrated better inhibition against α -amylase than those of flavanones. This could be due to the presence of a double bond at positions C-2 and C-3 in flavones and flavonols that might give an inhibition effect on the enzyme. In contrast with flavanones, this sub-class showed relatively lower inhibition. Subsequently, the tested compounds that showed ≥ 70% inhibition on α -amylase was further determined for their IC 50 values. Among natural flavones, increasing the number of -OH groups could increase the inhibition level. Chrysin ( F1 ) with two -OH groups on the A-ring only showed 22.59% inhibition. Nevertheless, the activity of the compound with another additional -OH group on the A-ring ( F4 ) was four-fold better than F1 . Even better activity could be reached when the compound contained the additional group on the B-ring, hence for compounds F2 (one additional -OH) and F3 (two additional -OH groups) exhibited IC 50 values of 13.18 and 2.18 µ M, respectively. This indicated that the -OH groups could be one of the factors that could increase the interaction number between the compound and enzyme leading to higher inhibition. The same phenomenon was also observed for natural flavonols, kaempferol ( F5 ) consisting of four -OH groups showed an IC 50 value of 10.43 µ M followed by quercetin ( F7 ) with five -OH groups displayed higher inhibition (IC 50 value 11.16 µ M). In contrast, the opposite effect appeared for morin ( F6 ), due to the different position of -OH groups ( meta ), the activity was weaker than in F7 . Furthermore, the results also displayed that luteolin ( F3 ) was better than quercetin ( F7 ) which indicated that the presence of the -OH group at 3-position on the C-ring was not essential for α -amylase inhibitory activity. This result was in line with a previous study which indicated that -OH groups at 4′- and 5′-position increased the inhibitory activity against α -amylase. 34 While natural flavanones and their brominated flavanones were found to be weak and not active agents toward α -amylase inhibitory activity whereas the percent inhibition was in the range of 12.85 to 51.13%. The introduction of bromine atoms into the flavonoid scaffold provided varied results. Among these tested compounds, F3a displayed the highest inhibition with an IC 50 value of 0.99 µ M, which was six-fold better than the reference drug acarbose (IC 50 value of 6.73 µ M). According to the findings, the structure-activity relationship of natural and brominated flavonoids could be rationalized into several important points as depicted in Fig. 5 . The Lineweaver-Burk plot was also established to illustrate the kinetic inhibition of α -amylase in the presence of F3a . The plot of 1/v vs 1/[CNP-G3] for F3a , showed that the straight lines intersected at the same point in the second quadrant, which specified that F3a was a mixed-type inhibitor (Fig. 6 ) in which F3a could bind with free enzymes, as well as enzyme-substrate complex to reduce the catalytic activity of α -amylase. Moreover, the equilibrium constants of binding of F3a to free enzyme ( K i ) and enzyme-substrate complex ( K is ) through plots of slope ( K m /V m ) and vertical intercept (1 /V m ) vs F3a with various concentrations were also determined. The value of K i was 0.18 µ M while K is values was 0.37 µ M suggesting that the affinity of F3a with free enzyme was higher than that with the enzyme-substrate complex. In vitro BSA-glycation mediated methyglyoxal (MG) inhibitory activity Increased glucose level is known as the typical indicator of DM, in which glucose as a reducing sugar is conducted to non-enzymatic glycation of many proteins including plasma protein, especially albumin. 14 Bovine serum albumin (BSA) was the glycation induced by a high reactive dicarbonyl species like methylglyoxal (MG), which reacted with proteins to form irreversible heterogeneous-AGEs. Since BSA may serve as a model protein for in vitro experiments, therefore in this study, the effect of tested compounds was applied to the in vitro BSA-glycation mediated by MG. Table 2 Inhibitory activity of natural flavonoids and the brominated flavonoids toward BSA-glycation mediated methylglyoxal (MG). Compound % Inhibition at 500 µ M* IC 50 ( µ M)* Flavones : Chrysin ( F1 ) 53.96 ± 2.71 n.d. 6,8-Dibromochrysin ( F1a ) 97.30 ± 0.19 50.90 ± 0.98 Apigenin ( F2 ) 73.66 ± 0.57 n.d. 6,8-Dibromoapigenin ( F2a ) 97.81 ± 2.08 57.01 ± 5.37 Luteolin ( F3 ) 96.32 ± 1.36 85.63 ± 4.97 6,8-Dibromoluteolin ( F3a ) 95.37 ± 1.42 79.48 ± 1.10 Baicalein ( F4 ) 75.66 ± 1.96 n.d. 8-Bromobaicalein ( F4a ) 36.68 ± 1.35 n.d. Flavonols : Kaempferol ( F5 ) 76.47 ± 1.05 n.d. 6,8-Dibromokaempferol ( F5a ) 72.24 ± 1.26 n.d. Morin ( F6 ) 94.69 ± 0.41 132.45 ± 2.47 6,8-Dibromomorin ( F6a ) 92.81 ± 0.92 61.22 ± 5.98 Quercetin ( F7 ) 100,00 ± 0.00 68.47 ± 0.74 6,8-Dibromoquercetin ( F7a ) 60.54 ± 2.14 n.d. Flavanones : Pinocembrin ( F8 ) 47.60 ± 3.64 n.d. 6,8-Dibromopinocembrin ( F8a ) 46.31 ± 2.23 n.d. Pinostrobin ( F9 ) 11.90 ± 3.63 n.d. 6,8-Dibromopinostrobin ( F9a ) 37.21 ± 7.62 n.d. Alpinetin ( F10 ) 45.65 ± 2.69 n.d. 6,8-Dibromoalpinetin ( F10a ) 56.89 ± 4.28 n.d. Naringenin ( F11 ) 83.98 ± 3.08 n.d. 6,8-Dibromonaringenin ( F11a ) 51.62 ± 2.03 64.51 ± 6.87 Hesperetin ( F12 ) 51.62 ± 5.42 n.d. 6,8-Dibromohesperetin ( F12a ) 63.27 ± 2.80 Aminoguanidine hydrochloride 79.50 ± 1.49 240.05 ± 5.02 *Mean ± Standard deviation from two independent experiments and each performed in triplicate. All selected flavonoids ( F1 – F12 ) and their brominated compounds ( F1a – F12a ) were evaluated for their anti-glycation potential. From the screening result at 500 µM of tested compounds in Table 2 , several flavonoids and brominated flavonoids significantly inhibited BSA glycation mediated by MG which showed better inhibition (93.40 to 99.75%) compared with its positive control, aminoguanidine hydrochloride (79.66%). Flavones and their brominated gave the inhibition in the range of 35.56–97.82%, while the inhibition ranging from 60.85 to 99.75% was shown by flavonols and their brominated derivatives. In addition, flavanones and their brominated compounds displayed inhibition in the range of 11.90–96.53%. Among natural flavones, the increasing percent inhibition was observed by increasing the number -of OH groups with the order: chrysin ( F1 ) < apigenin ( F2 ) < baicalein ( F4 ) < luteolin ( F3 ). This phenomenon indicated that the -OH groups played a crucial role in inhibiting the glycation process because they increased the number of intermolecular hydrogen bonds whereas the lower energy barriers were accompanied by more H-bonds. 37 Moreover, the C = O group of the C-ring facilitated the intermolecular H-bond formation, in which the flavonoid could effectively hinder the AGEs formation. 37 In addition, previously, it has been reported that the -OH group at the 5-position on the A-ring was essential for trapping MG by flavonoids. 38 Furthermore, two brominated flavones ( F1a and F2a ) exhibited better activities than their parent compounds ( F1 and F2 ). This implied that the presence of bromine atoms on the A-ring gave the advantage effect for the activity due to the fact that halogens could act as hydrogen bond acceptors and potentially make a more favorable contribution to the binding. In contrast, the observed F4a containing only one bromine atom on the A-ring at 8-position exhibited lower inhibition compared with the starting material ( F4 ). This described that the presence of bromine atom at 8-position did not give a beneficial effect on the activity. The introduction of bromine atom at 6-position on the A-ring of flavone could enhance the inhibition of the glycation process. However, this outcome only occurred for flavone containing two -OH groups on the A-ring and one additional -OH group on the B-ring. For example, 6,8-dibromoluteolin ( F3a ) with two more additional -OH groups on the B-ring displayed no increased effect compared with its parent compound luteolin ( F3 ). This specified that the inhibition was favored when the inhibitor was not a bulky compound that may offer steric hindrance. In the case of natural flavonols, the same trend of the number -OH groups were also observed which the percent inhibition of glycation follows the order: kaempferol ( F5 ) < morin ( F6 ) < quercetin ( F7 ). Albeit F6 and F7 contained the same number of -OH groups, due to the different positions, the activity showed a slight dissimilar effect. This denoted that the catechol moiety on the B-ring was favored over the meta -OH. In addition, the introduction of bromine atoms at 6- and 8- positions of flavonols ( F5a – F7a ) exhibited a decrease in inhibition compared with their starting materials. This due to the bulky compounds were generated from this series of derivatives leading to the difficulty to access the inhibition. For flavanones, the importance of the -OH group at the 5-position of flavonoids was also observed when investigated pinocembrin ( F8 ) containing two -OH group at 5,7-positions and pinostrobin ( F9 ) with one -OH group at 7-position and one -OMe group at 5-position, displayed a significant difference in the inhibition. The same result was also observed when the 7-OH was replaced by 7-OMe which was possessed by alpinetin ( F10 ), which revealed that the -OH group at the 7-position played a crucial role in inhibiting the glycation process. Furthermore, the increase of the number -OH groups in flavanones was also essential for the activity, for instance, pinocembrin ( F8 ) contained two -OH groups on the A-ring with inhibition of 47.60%, then underwent slightly increase inhibition for naringenin ( F11 ) which contained one more additional -OH group on the B-ring with inhibition of 56.89%. But then slightly reduce the inhibition level once the -OH group at 4-position in the B-ring was replaced by -OMe, even though compound F12 contained one more -OH group at 3′-position on the B-ring. This revealed that the -OH group at the 4′-position in the B-ring of flavanone was favored over the 3′-position. As stated in a previous study that the presence of one -OH and one -OMe group showed varied activity depending upon the position of the -OH substituent, whereas a compound with an ortho hydroxy was found to be a not good inhibitor. 39 Subsequently, several selected compounds that exhibited percent inhibition ≥ 80% were further investigated for their IC 50 values by varying the concentrations as depicted in Table 2 . The criteria of IC 50 value is categorized as follow: IC 50 500 µ M (not active). According to the results, 6,8-dibromochrysin ( F1a ) showed the highest inhibition with an IC 50 value of 50.90 µ M, followed by compounds F2a (IC 50 = 57.01 µ M), F6a (IC 50 = 61.22 µM), F11a (IC 50 = 64.51 µ M), F7 (IC 50 = 68.47 µ M), F3a (IC 50 = 79.48 µ M), and F3 (IC 50 = 85.6 µ M), in which these compounds displayed strong inhibition toward the BSA-glycation process mediated MG. It seems that the anti-glycation activity favored the compound that less bulky. Moreover, morin ( F6 ) showed a relatively strong effect as an anti-glycation agent with an IC 50 value of 132.45 µ M. All these selected compounds mainly showed 2- to 6-fold better activities compared with its standard drug, aminoguanidine hydrochloride (IC 50 = 240.05 µ M). Materials and Methods Materials Plant Materials Two plant materials were used in this work including the rhizomes of Boesenbergia rotunda (L.) Mansf. which was purchased from the herbal drug store in Bangkok-Thailand, and the flowers of Impatiens balsamina L. was collected from Indonesia. Equipment and Instruments 1 H NMR (500 MHz) and 13 C NMR (125 MHz) spectra were recorded with a JEOL spectrometer (JNM-ECZ500R/S1) while 1 H NMR (400 MHz) and 13 C NMR (100 MHz) spectra were recorded on a Bruker 400 AVANCE spectrometer, and chemical shifts were recorded in parts per million (ppm) and coupling constants ( J ) were given in Hertz. The LC-QTOF-MS/MS analysis was performed on an Agilent HPLC 1260 series coupled with a QTOF 6540 UHD accurate mass (Agilent Technologies, Waldbronn, Germany). The fluorescence intensity was measured by the EnSight Multimode Plate Reader PerkinElmer. Chemicals Chrysin, apigenin, luteolin, baicalein, quercetin hydrate, naringenin, and hesperetin were purchased from Tokyo Chemical Industry company, while morin was bought from Fluka and used without further purification. Other synthetic reagents were purchased from Sigma-Aldrich company or otherwise stated. All solvents used in this study were purified by standard methods, except for those which were reagent grades. The progress of the reaction was monitored by Thin Layer Chromatography (TLC) using TLC silica gel 60 F 254 Merck. Purification of semisynthetic compounds was performed by column chromatography using silica gel (70–230 mesh) of SiliaFlash® G60 (Canada). α -Glucosidase (Sigma G5003) derived from Baker’s yeast, α -amylase from porcine pancreas type VI-B, 4-nitrophenyl α -D-glucopyranoside ( p- NPG, Sigma N1377), 2-chloro-4-nitrophenyl α -D-maltotrioside (CNP-G3), methylglyoxal solution, acarbose, and bovine serum albumin (BSA) were purchased from Sigma-Aldrich. Aminoguanidine hydrochloride was used as a positive control for anti-glycation and was purchased from Tokyo Chemical Industry company. Methods Isolation of kaempferol (F5) from Impatiens balsamina L. flowers The dried and powdered Impatiens balsamina L. flowers (272 g) was done by maceration in MeOH for three days at room temperature, filtered, and concentrated by a rotary evaporator. This step was repeated three times to obtain the dark brown MeOH crude extract (97.62 g, 36%). This crude extract was then partitioned with EtOAc and H 2 O and separated. EtOAc fraction was then concentrated to obtain 38.82 g of EtOAc extract. Subsequently, this fraction was subjected to silica gel column which was initially eluted with hexane-EtOAc and EtOAc-MeOH by increasing polarity to give five fractions (1–5). The precipitate from the fraction 1 was then collected and washed with hexane:EtOAc (1:1) to give kaempferol ( F5 ) as yellow powder (92 mg). Fraction 2 (1.46 g) was then further purified by using silica gel column and eluted with hexane-EtOAc to give six subfractions (2a–2f). Subfractions 2d and 2e formed precipitates. Washing the precipitate using hexane:EtOAc (1:1) gave 1.0 g of kaempferol ( F5 ). Kaempferol ( F5 ): yellow powder (0.4%), 1 H NMR (400 MHz, DMSO- d 6 ) δ (ppm) 12.47 (s, 5-OH), 10.80 (s, 3-OH), 10.12 (s, 7-OH), 9.37 (s, 4′-OH), 8.04 (d, J = 8.8 Hz, 2H), 6.92 (d, J = 6.8 Hz, 2H), and 6.19 (d, J = 2.4 Hz, 1H); 13 C NMR (100 MHz, DMSO- d 6 ) δ (ppm) 175.9, 163.9, 160.7, 159.2, 156.2, 146.9, 135.7, 129.5, 121.7, 115.5, 103.1, 98.2, and 93.5. Isolation of pinocembrin (F8), pinostrobin (F9) and alpinetin (F10) from Boesenbergia rotunda (L.) Mansf. rhizomes The extraction of the dried and powdered Boesenbergia rotunda (L.) Mansf. rhizomes (5.0 kg) was done by maceration in MeOH for three days at room temperature, filtered, and concentrated by a rotary evaporator. This step was repeated three times to obtain the dark brown MeOH crude extract (700 g, 14%). This crude extract was then subjected to silica gel column which was initially eluted with hexane-EtOAc and EtOAc-MeOH by increasing polarity to give seven fractions. Washing the precipitate in fraction 5 using EtOAc gave pinocembrin ( F8 ) as pale-yellow solid 113 g (16%). After recrystallization of precipitates from fractions 2, 3, and 4 with hexane and EtOAc, pinostrobin ( F9 ) as colorless crystal 320 g (46%) was attained. In addition, recrystallization of the precipitate in fraction 6 using CH 2 Cl 2 and MeOH, furnished alpinetin ( F10 ) as a pale-yellow solid 57 g (8.1%). Pinocembrin ( F8 ): pale-yellow solid (16%), 1 H NMR (500 MHz, Acetone- d 6 ) δ (ppm) 12.16 (s, 5-OH), 9.76 (s, 7-OH), 7.57 (dd, J = 7.0, 1.5 Hz, 2H), 7.42 (m, 3H), 6.00 (d, J = 2.5 Hz, 1H), 5.97 (d, J = 2.0 Hz, 1H), 5.57 (dd, J = 13.0, 3.5 Hz, 1H), 3.17 (dd, J = 17.0, 12.5 Hz, 1H), and 2.81 (dd, J = 17.0, 3.0 Hz, 1H); 13 C NMR (125 MHz, Acetone- d6 ) δ (ppm) 196.8, 167.4, 165.2, 164.1, 140.0, 129.4, 127.3, 103.2, 96.9, 95.9, 79.9, and 43.5. Pinostrobin ( F9 ): colorless crystal (46%), 1 H NMR (500 MHz, CDCl 3 ) δ (ppm) 12.02 (s, 5-OH), 7.42 (m, 5H), 6.08 (d, J = 2.5 Hz, 1H), 6.07 (d, J = 2.0 Hz, 1H), 5.42 (dd, J = 13.5, 3.5 Hz, 1H), 3.81 (s, 3H), 3.09 (dd, J = 17.0, 13.0 Hz, 1H), and 2.83 (dd, J = 17.0, 3.0 Hz, 1H); 13 C NMR (125 MHz, CDCl 3 ) δ (ppm) 196.3, 168.5, 164.3, 162.9, 138.5, 129.0, 126.3, 103.3, 95.3, 94.4, 79.4, 55.8, and 43.5. Alpinetin ( F10 ): pale-yellow solid (8.1%), 1 H NMR (500 MHz, DMSO- d 6 ) δ (ppm) 10.57 (s, 7-OH), 7.49 (dd, J = 6.5, 1.5 Hz, 2H), 7.39 (m, 3H), 6.04 (dd, J = 35.0, 2.0 Hz, 1H), 5.48 (dd, J = 12.5, 3.0 Hz, 1H), 3.74 (s, 3H), 2.98 (dd, J = 16.5, 12.5 Hz, 1H), and 2.62 (dd, J = 16.5, 3.5 Hz, 1H); 13 C NMR (125 MHz, DMSO- d 6 ) δ (ppm) 187.4, 164.4, 164.1, 162.2, 139.2, 128.5, 128.3, 126.5, 104.5, 95.7, 93.4. 78.1, 55.6, and 44.9. Synthesis of bromoflavones Bromination of chrysin, apigenin and luteolin was performed according to the previously method. 29 6,8-Dibromochrysin ( F1a ) was obtained by reacting Chrysin ( F1 ,1 mmol) in acetone:water 5:1 and NaBr (3 mmol). After cooling, oxone (3 mmol) was added and the mixture was stirred at room temperature for 3 h. The final solution was treated with Na 2 S 2 O 3 and evaporated under reduced pressure. The residue was recrystallized from MeOH to yield 92% yellow powder as a brominated product ( F1a ). The same procedure was applied for synthesizing 6,8-dibromoapigenin ( F2a ) and 6,8-dibromoluteolin ( F3a ). While the brominated baicalein ( F4a ) was obtained by mixing 1 mmol baicalein (F4) and 1 mmol N -bromosuccinimide (NBS) in 4.0 mL tetrahydrofuran (THF) in the presence of 5.0 µ L concentrated H 2 SO 4 as described by a previously method. 31 The reaction mixture was stirred at room temperature for 12 h before extraction with EtOAc. The precipitated product was washed with 10% aqueous NaHSO 4 solution, dried over anhydrous Na 2 SO 4 , and concentrated under reduced pressure. The residue was recrystallized from MeOH to the target compound (58%) as a yellow powder. 6,8-Dibromochrysin ( F1a ): yellow powder (92% yield). 1 H NMR (500 MHz, DMSO- d 6 ) δ (ppm) 8.10 (d, J = 7.5 Hz, 2H), 7.60 (m, 3H), and 7.14 (s, 1H); 13 C NMR (125 MHz, DMSO- d 6 ) δ (ppm) 181.6, 163.5, 157.5, 157.1, 152.3, 132.6, 130.3, 129.3, 126.5, 105.2, 105.1, 94.6, and 88.5. 6,8-Dibromoapigenin ( F2a ): yellow powder (95% yield). 1 H NMR (500 MHz, DMSO- d 6 ) δ (ppm) 8.00 (d, J = 9.0 Hz, 2H), 6.98 (s, 1H), and 7.95 (m, 2H); 13 C NMR (125 MHz, DMSO- d 6 ) δ (ppm) 181.5, 164.3, 161.8, 157.2, 157.1, 152.2, 128.8, 120.8, 116.3, 116.1, 104.9, 94.4, and 88.4. 6,8-Dibromoluteolin ( F3a ): yellow powder (97% yield). 1 H NMR (500 MHz, DMSO- d 6 ) δ (ppm) 7.52 (d, J = 10 Hz, 2H), 6.92 (d, J = 8 Hz, 1H), and and 6.89 (s, 1H); 13 C NMR (125 MHz, DMSO- d 6 ) δ (ppm) 181.3, 164.4, 157.4, 157.1, 152.2, 150.3, 145,9, 121.1, 119.4, 116.1, 113.7, 104.7, 102.7, 94.5, and 88.4. HRMS m/z (ESI+): calculated for C 15 H 8 Br 2 O 6 ([M + H] + ): 442.8766, found 442.8756. 8-Bromobaicalein ( F4a ): yellow powder (58% yield). 1 H NMR (500 MHz, DMS0- d6 ) δ (ppm) 8.12 (d, J = 9.5 Hz, 2H), 7.61 (m, 3H), and 7.06 (s, 1H); 13 C NMR (125 MHz, DMSO- d 6 ) δ (ppm) 182.2, 163.0, 151.6, 146.7, 146.4, 132.3, 130.7, 129.7, 129.4, 126.4, 104.7, and 87.0. Synthesis of bromoflavonols The preparation of 6,8-dibromokaempferol ( F5a ) and 6,8-dibromomorin ( F6a ) were performed by using a previously method. 29 By mixing the flavonol (1 mmol) with NaBr (3 mmol) and oxone (3 mmol) in acetone and H 2 O (5:1) for 3 hours. Then, the reaction was stopped by pouring the mixture into H 2 O and treating with Na 2 S 2 O 3 . The filtration was carried out and washed the residue with H 2 O. Recrystallized the residue with MeOH to get the desired product. While 6,8-dibromoquercetin ( F7a ) was obtained as described by a previously method. 32 The target compound was synthesized by mixing quercetin ( F7 , 1 mmol) with diluted Br 2 (1 mL in 5 mL CH 3 COOH) at 35 o C for 3 days. Filtered the precipitate, washed with H 2 O, and recrystallized from MeOH to get the product. 6,8-Dibromokaempferol ( F5a ): light yellow powder (34% yield). 1 H NMR (500 MHz, DMSO- d 6 ) δ 13.41 (s, 5-OH), 10.29 (s, 3-OH), 9.88 (s, 7-OH), 8.16 (d, J = 8.5 Hz, 2H), and 6.96 (d, J = 9.0 Hz, 2H); 13 C NMR (125 MHz, DMSO- d 6 ) δ (ppm) 175.5, 159.8, 156.9, 156.4, 150.9, 147.8, 136.1, 129.8, 121.6, 115.8, 104.3, 93.7, and 88.0. 6,8-Dibromomorin ( F6a ): light yellow powder (53% yield). 1 H NMR (500 MHz, DMSO- d 6 ) δ 7.53 (s, 1H), 6.64 (s, 1H), and 6.40 (s, 1H); 13 C NMR (125 MHz, DMSO- d 6 ) δ (ppm) 176.2, 160.6, 159.7, 156.8, 156.4, 153.0, 147.7, 136.9, 134.1, 110.7, 104.4, 103.9, 98.6, 98.2, and 86.0. 6,8-Dibromoquercetin ( F7a ): light yellow powder (36% yield). 1 H NMR (500 MHz, Acetone- d 6 ) δ (ppm) 7.32, (d J = 2.0 Hz, 1H), 7.19 (dd, J = 8.0, 2.0 Hz, 1H), and 6.89 (d, J = 8.0 Hz, 1H); 13 C NMR (125 MHz, Acetone- d 6 ) δ (ppm) 195.5, 160.0, 159.6, 154.6, 147.0, 145.1, 124.8, 121.9, 117.3, 115.2, 108.9, 101.6, 91.7, and 90.9. Synthesis of bromoflavanones Flavanone (1 mmol) was mixed with acetone and H 2 O (5:1), then NaBr (3 mmol) was added and stirred for 5 minutes. Subsequently, oxone (3 mmol) was added to the mixture solution and stirred at room temperature for 3 h. The mixture was poured into H 2 O, then treated with Na 2 S 2 O 3 and filtered. The residue was then washed with H 2 O. Recrystallized the residue with MeOH to obtain the target compound. 6,8-Dibromopinocembrin ( F8a ): white powder (69% yield). 1 H NMR (500 MHz, DMSO- d 6 ) δ 7.54 (d, J = 7.5 Hz, 2H), 7.43 (m, 3H), 5.78 (dd, J = 12.0, 3.0 Hz, 1H), 3.38 (dd, J = 17.0, 12.0 Hz, 1H), and 2.99 (dd, J = 17.0, 3.0 Hz, 1H); 13 C NMR (125 MHz, DMSO- d 6 ) δ 196.5, 159.3, 158.5, 157.7, 138.2, 128.8, 126.5, 102.9, 91.0, 90.0, 79.2, 79.1, and 41.2. 6,8-Dibromopinostrobin ( F9a ): yellow powder (75% yield). 1 H NMR (500 MHz, CDCl 3 ) δ 7.46 (m, 5H), 5.59 (dd, J = 12.5, 3.5 Hz, 1H), 3.96 (s, 3H), 3.17 (dd, J = 17.0, 3.5 Hz, 1H), and 3.04 (dd, J = 17.0, 3.5 Hz, 1H); 13 C NMR (125 MHz, CDCl 3 ) δ 196.7, 162.5, 159.3, 158.1, 137.5, 129.2, 129.1, 126.0. 106.3, 98.7, 97.0, 79.6, 61.1, and 42.8. 6,8-Dibromoalpinetin ( F10a ): white powder (87% yield). 1 H NMR (500 MHz, DMSO- d 6 ) δ 7.54 (d, J = 7.0 Hz, 2H), 7.43 (m, 3H), 5.74 (dd, J = 12.5, 3.5 Hz, 2H), 3.76 (s, 3H), 3.16 (dd, J = 16.5, 12.5 Hz, 1H), and 2.85 (dd, J = 17.0, 3.5 Hz, 2H); 13 C NMR (125 MHz, DMSO- d 6 ) δ 187.6, 158.8, 157.4, 157.3, 138.5, 128.7, 128.6, 126.3, 109.8, 101.1, 95.7, 78.8, 61.1, and 43.8. HRMS m/z (ESI+): calculated for C 16 H 12 Br 2 O 4 ([M + H] + ): 426.9181, found 426.9170. 6,8-Dibromonaringenin ( F11a ): white powder (65% yield). 1 H NMR (500 MHz, DMSO- d 6 ) δ 7.34 (d, J = 9.0 Hz, 2H), 6.81 (d, J = 9.0 Hz, 1H), 5.62 (dd, J = 13.0, 3.5 Hz, 1H), 3.39 (dd, J = 17.0, 12.0 Hz, 1H), and 2.88 (dd, J = 17.0, 3.0 Hz, 1H); 13 C NMR (DMSO- d 6 ) δ 197.0, 159.2, 158.5, 158.0, 157.9, 128.3, 128.3, 115.4, 102.9, 90.8. 89.9, 79.3, and 41.2. 6,8-Dibromohesperetin ( F12a ): white powder (98% yield). 1 H NMR (500 MHz, DMSO- d 6 ) δ 6.95 (d, J = 8.5 Hz, 2H), 6.89 (dd, J = 8.5, 2.5 Hz, 1H), 5.61 (dd, J = 12.0, 3.0 Hz, 1H), 3.87 (s, 3H), 3.33 (dd, J = 17.0, 12.0 Hz, 2H), and 2.90 (dd, J = 17.0, 8.5 Hz, 1H); 13 C NMR (125 MHz, DMSO- d 6 ) δ 196.9, 159.3, 158.6, 157.8, 148.2, 146.6, 130.5, 117.9, 114.1, 112.1, 102.9, 90.9, 90.0, 79.2, 55.8, and 41.3. In vitro α-glucosidase inhibitory activity The determination of α -glucosidase inhibitory of selected natural flavonoids and their brominated was performed according to the previously described protocol. 29 The stock solution of tested compounds and acarbose were dissolved in DMSO and further diluted with phosphate buffer pH 6.9, α -glucosidase and the substrate p -NPG were both dissolved in phosphate buffer pH 6.9. About 10 µ L of the tested compound was inserted into a well of 96-wells microplate followed by adding 40 µ L of α -glucosidase which was then preincubated at 37 o C for 10 minutes by shaking at 500 rpm. Subsequently, 50 µ L of substrate p -NPG was added, and the incubation was continued at 37 o C for 30 minutes by shaking at 500 rpm. Finally, the reaction was terminated by the addition of 100 µ L of 1 M Na 2 CO 3 . The absorbance was measured at 405 nm using a microplate reader. Inhibitory activity was calculated by Eq. 1. Where A 0 was the absorbance of blank (contained the same volume of the buffer solution instead of the tested compound); A t was the absorbance of the reaction in the presence of a tested compound, α -glucosidase, and substrate p- NPG. The IC 50 values were defined as the concentration of an inhibitor required to inhibit 50% of the α -glucosidase activity under the assay conditions. The inhibition assay was performed in triplicate and in two independent experiments for all tested compounds. In vitro α-amylase inhibitory activity The ⍺-amylase assay was performed in 96-well microplates using a final volume of 200 µ L as the previous method with some modification. 35 , 36 Unless otherwise stated, experiments were performed with phosphate buffer (0.1 mM) containing 0.02% NaN 3 and adjusted to pH 6.0 with 2.0 M H 3 PO 4 . Stock solutions of α -amylase and CNP-G3 in phosphate buffer were prepared at concentrations of 1 mg/mL and 0.5 mM, respectively. Enzymatic reaction mixtures consisted of test compound (10 µ L), potassium phosphate buffer (140 µ L), an enzyme (20 µ L), then preincubated in phosphate buffer at 37 o C for 10 minutes by shaking at 500 rpm. Subsequently, the substrate (30 µ L) was incubated at 37° C for 30 minutes. Enzymatic activity was detected by spectrophotometry at 405 nm. Inhibitory activity was calculated by Eq. 1. The IC 50 values were defined as the concentration of an inhibitor required to inhibit 50% of the α -amylase activity under the assay conditions. The IC 50 value was further determined for the tested samples that show the inhibition ≥ 70%. For all tested compounds, the inhibition assay was performed in triplicate and two-independent experiments. Inhibitory kinetic analysis of selected compounds against enzymes The inhibition types of 8-bromobaicalein ( F4a ) towards α -glucosidase and 6,8-dibromoluteolin ( F3a ) towards α -amylase were determined from Lineweaver-Burk plots. Typically, three different concentrations of each compound around the IC 50 value were chosen. The inhibition type was determined using various concentrations of p -NPG substrate for α -glucosidase and CNP-G3 for α -amylase. Anti-glycation activity In brief, Bovine Serum Albumin solution (10 mg/mL) was prepared in 0.1 M of phosphate buffer pH 7.4 containing 0.02% NaN 3 . In addition, 14 mM methylglyoxal (MG) was prepared in a phosphate buffer. The test compound and standard inhibitor were prepared in dimethyl sulfoxide (DMSO). About 20 µ L of inhibitor, 80 µ L of phosphate buffer, and 50 µ L of 14 mM MG were mixed in the 96-well and then incubated at 37 o C for 2 h. After preincubation, 50 µ L of BSA was added to initiate the reaction. The reaction mixture was then incubated at 37 o C for 1 day. After incubation, each sample was examined for the development of specific fluorescence (excitation 370 nm; emission 450 nm) against a blank (without inhibitor) on a microplate reader and then calculated as Eq. 2. Afterward, the IC 50 value was further investigated for the tested compounds that show inhibition ≥ 80%. For all tested compounds, the inhibition assay was performed in triplicate. Statistical Analysis. All the experiments were carried out in triplicate and in two independent experiments. The inhibition percentage and or IC 50 values were calculated by using Microsoft Excel version 16.64 and a GraphPad Prism version 9. The data are expressed as means of two independent experiments ± standard deviations. c Log P values were obtained from ChemDraw Professional 16.0. Declarations Acknowledgements We appreciate Chulalongkorn University–Graduate Programme Scholarship for ASEAN and NON-ASEAN Countries Academic Year 2019 for supporting Ms. Rita Hairani to study at Chulalongkorn University. Ms. R. Hairani is also grateful to Graduate School Thesis Grant, Chulalongkorn University for financial support. Author contributions statement R.H. performed all experiments including preparation of all compounds, in vitro ⍺ -glucosidase and ⍺ -amylase inhibitory activity assay, as well as anti-glycation activity. W.C. designed the synthesis of bromoflavonoids, supervised the study, provided critical discussion, and prepared the manuscript to be published. All authors have read and agreed to the published version of the manuscript. Competing interest The authors declare no conflict of interest. Data Availability The data supporting the findings of this study are available within the paper and its Supplementary Information files. References Rammohan, A., Bhaskar, B. 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Flavones from Combretum quadrangulare growing in Vietnam and their alpha-glucosidase inhibitory activity. Molecules 26 , 2531. https://doi.org/10.3390/molecules26092531 (2021). Napolitano, J. G., Lankin, D. C., Chen, S. N. & Pauli, G. F. Complete 1 H NMR spectral analysis of ten chemical markers of Ginkgo biloba . Magn. Reson. Chem. 50 , 569–575. https://doi.org/10.1002/mrc.3829 (2012). Poungcho, P., Hairani, R., Chaotham, C., De-Eknamkul, W. & Chavasiri, W. Methoxylated chrysin and quercetin as potent stimulators of melanogenesis. Int. J. Mol. Sci. 26 , 3281. https://doi.org/10.3390/ijms26073281 (2025). Hairani, R. & Chavasiri, W. A new series of chrysin derivatives as potent non-saccharide 훂-glucosidase inhibitor. Fitoterapia 163 , 105301. https://doi.org/10.1016/j.fitote.2022.105301 (2022). Li, Y., Cai, S., He, K. & Wang, Q. Semisynthesis of polymethoxyflavonoids from naringin and hesperidin. J. Chem. Res. 38 , 287–290. https://doi.org/10.3184/174751914X13966139490181 (2014). Boonyasuppayakorn, S. et al. The 8-bromobaicalein inhibited the replication of dengue, and Zika viruses and targeted the dengue polymerase. Sci. Rep. 13 , 4891. https://doi.org/10.1038/s41598-023-32049-x (2023). Peng, M., Liu, F., Feng, X., Yang, F. & Yang, X. Synthesis of brominated quercetin derivatives using distinct brominating systems. Asian J. Chem. 26 , 4701–4703. https://doi.org/10.14233/ajchem.2014.16175 (2014). Şöhretoğlu, D. & Sari, S. Flavonoids as alpha-glucosidase inhibitors: mechanistic approaches merged with enzyme kinetics and molecular modelling. Phytochem. Rev. 19 , 1081–1092. https://doi.org/10.1007/s11101-019-09610-6 (2020). Zhao, Y., Wang, M. & Huang, G. Structure-activity relationship and interaction mechanism of nine structurally similar flavonoids and α-amylase. J. Funct. Foods. 86 , 104739. https://doi.org/10.1016/j.jff.2021.104739 (2021). Okutan, L., Kongstad, K. T., Jager, A. K. & Staerk, D. High-resolution alpha-amylase assay combined with high-performance liquid chromatography-solid-phase extraction-nuclear magnetic resonance spectroscopy for expedited identification of alpha-amylase inhibitors: proof of concept and alpha-amylase inhibitor in cinnamon. J. Agric. Food Chem. 62 , 11465–11471. https://doi.org/10.1021/jf5047283 (2014). Sun, H. et al. Natural prenylchalconaringenins and prenylnaringenins as antidiabetic agents: Alpha-glucosidase and alpha-amylase inhibition and in vivo antihyperglycemic and antihyperlipidemic effects. J. Agric. Food Chem. 65 , 1574–1581. https://doi.org/10.1021/acs.jafc.6b05445 (2017). Rezazadeh, S., Ebrahimi, A. & Nowroozi, A. The effects of structural properties on the methylglyoxal scavenging mechanism of flavonoid aglycones: A quantum mechanical study. Comput. Theor. Chem. 1118 , 26–38. https://doi.org/10.1016/j.comptc.2017.09.001 (2017). Shao, X. et al. Essential structural requirements and additive effects for flavonoids to scavenge methylglyoxal. J. Agric. Food Chem. 62 , 3202–3210. https://doi.org/10.1021/jf500204s (2014). Taha, M. et al. Synthesis of 4-methoxybenzoylhydrazones and evaluation of their antiglycation activity. Molecules 19 , 1286–1301. https://doi.org/10.3390/molecules19011286 (2014). Additional Declarations No competing interests reported. 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Chavasiri","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7UlEQVRIiWNgGAWjYJCCA2AEAh+ADD6oaAJ+LQkQLYwzgGw2iKABXi0MMC3MPMRoMTh+9uHhwh935MxnNzB+tqm5k9jGfoDxww+GP3k4tZxJNzg8I+GZscydA8zSOceeJbbxJDBL9jAYFOPSYnYgjeEwT8LhxBkSCWzMuQ2HE9uAHpcGmpXYgEvL+WdIWixBWvgfMP/Gq+UGsi2MIC1ABl5b7G+AbEk7bCwhkdgs2XPssHGbxMM2yx4DY5xaJPvTmD/z2ByWk5BIPvjhR81h2X7+5MM3flTI4dSCBBgbkBgGhNWPglEwCkbBKMANAClvWZEzwnPqAAAAAElFTkSuQmCC","orcid":"","institution":"Chulalongkorn University","correspondingAuthor":true,"prefix":"","firstName":"Warinthorn","middleName":"","lastName":"Chavasiri","suffix":""}],"badges":[],"createdAt":"2025-04-15 08:53:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6452882/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6452882/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-09040-9","type":"published","date":"2025-07-15T15:57:06+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81706547,"identity":"7e4f847a-593d-4ac0-a9ea-fef155efefe6","added_by":"auto","created_at":"2025-04-30 13:49:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":58266,"visible":true,"origin":"","legend":"\u003cp\u003eTwelve selected natural flavonoids.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6452882/v1/8a200f12c92e2ac9f57a30f8.png"},{"id":81706548,"identity":"1d25ce69-7c94-49a4-8fad-0f9a0ad98a08","added_by":"auto","created_at":"2025-04-30 13:49:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":81218,"visible":true,"origin":"","legend":"\u003cp\u003eSynthesis of bromoflavonoids\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6452882/v1/1533967065f4fe237b21398f.png"},{"id":81707594,"identity":"990be401-6051-4ef6-9e9f-ffb4e7c09e6f","added_by":"auto","created_at":"2025-04-30 13:57:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":127801,"visible":true,"origin":"","legend":"\u003cp\u003eStructure-activity relationship natural and brominated flavonoids of flavones (A) and flavonols (B) on \u003cem\u003ea\u003c/em\u003e-glucosidase.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6452882/v1/fff6c520f7544c205e411eee.png"},{"id":81707595,"identity":"29f13d56-32bf-4011-8d16-c52db93fe187","added_by":"auto","created_at":"2025-04-30 13:57:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":43647,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Lineweaver-Burk Plot of F4a towards \u003cem\u003ea\u003c/em\u003e-glucosidase (B) Plots of slope versus concentration of \u003cstrong\u003eF4a\u003c/strong\u003e for determining the inhibition constant \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e (C) Plots of intercept versus concentration of \u003cstrong\u003eF4a\u003c/strong\u003e for determining the inhibition constant \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eis\u003c/em\u003e\u003c/sub\u003e\u003csub\u003e.\u003c/sub\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6452882/v1/6840199f6d11e6caa4b0dc3b.png"},{"id":81706550,"identity":"f510ca83-76af-4ba4-8dfe-f3081a8280bb","added_by":"auto","created_at":"2025-04-30 13:49:42","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":45800,"visible":true,"origin":"","legend":"\u003cp\u003eStructure-activity relationship natural and brominated flavonoids on \u003cem\u003ea\u003c/em\u003e-amylase.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6452882/v1/3d03f22fca2d07e2b96491dd.png"},{"id":81706552,"identity":"56b4cf66-87d0-45d1-9ddb-a84334dfa8d9","added_by":"auto","created_at":"2025-04-30 13:49:42","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":44374,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Lineweaver-Burk Plot of \u003cstrong\u003eF3a\u003c/strong\u003e towards \u003cem\u003ea\u003c/em\u003e-Amylase (B) Plots of slope versus concentration of \u003cstrong\u003eF3a\u003c/strong\u003e for determining the inhibition constant \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e (C) Plots of intercept versus concentration of \u003cstrong\u003eF3a\u003c/strong\u003e for determining the inhibition constant \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eis\u003c/em\u003e\u003c/sub\u003e.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6452882/v1/c078968e5adaac9acf3c6853.png"},{"id":87220330,"identity":"81b19707-1a26-4da0-9886-ffb763dd570d","added_by":"auto","created_at":"2025-07-21 16:11:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2038735,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6452882/v1/d14a739e-f596-484f-82b6-b1fc379da500.pdf"},{"id":81706562,"identity":"8a4b617f-e4a0-426b-8497-306f49e56165","added_by":"auto","created_at":"2025-04-30 13:49:42","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":3275833,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformationScientificReports.docx","url":"https://assets-eu.researchsquare.com/files/rs-6452882/v1/425995d1d549dfdf09e2a7fa.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Synthesis of promising brominated flavonoids as antidiabetic and anti-glycation agents","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDiabetes mellitus (DM), a chronic metabolic disorder with a high global prevalence, is divided into two types namely type 1 DM which is generated by dysfunction of \u003cem\u003eβ\u003c/em\u003e-pancreatic cells, and type 2 DM which is caused by impairment of insulin secretion due to insulin resistance and results in disturbances in glucose homeostasis.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e Type 2 DM is recognized as a major threat to human health and development and has emerged as one of the most prevalent public health issues.\u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e In the case of type 2 DM, the condition could lead to an increase in blood glucose levels. The inhibition of the activities of \u003cem\u003eα\u003c/em\u003e-glucosidase and \u003cem\u003eα\u003c/em\u003e-amylase enzymes, which catalyze carbohydrate hydrolysis, is believed as one approach for controlling blood glucose levels.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eOver the last two decades, the demand for antidiabetic medications has risen quickly as DM becomes more prevalent.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e One of the drugs used to treat type 2 DM and known to inhibit \u003cem\u003eα\u003c/em\u003e-glucosidase and \u003cem\u003eα\u003c/em\u003e-amylase activities, \u003cem\u003ei.e.\u003c/em\u003e acarbose has some drawbacks including the side effects related to diarrhea and flatulence.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e Moreover, its production still faces significant challenges that drive up manufacturing costs, one of which is through genetic engineering.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eOn the other hand, diabetic patients tend to develop vascular complications, which are a leading cause of morbidity and mortality.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e Chronic hyperglycemia resulting in advanced glycation end-products (AGEs) facilitates diabetes-related complications, such as diabetic retinopathy, nephropathy, peripheral neuropathy, atherosclerosis, and other complications.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e The primary serum protein, albumin, has a number of roles and is very susceptible to several environmental factors, with glucose being one of the most significant.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e During long-standing hyperglycemia states in DM, the steadily increasing glucose level tends to form covalent adducts with plasma proteins via a non-enzymatic process named glycation or Maillard reaction, resulting in the formation of AGEs.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eA class of compounds has been identified either to prevent the formation or to degrade existing AGEs, which have been manufactured and patented.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e Aminoguanidine, known as the first AGEs inhibitor, could trap or scavenge the reactive carbonyl intermediates such as glyoxal and methylglyoxal (MG) in the glycation process.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e However, a previous study revealed that the use of a high concentration of aminoguanidine was required to trap the reactive species due to its half-life in plasma is short.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e This is one of the disadvantages of aminoguanidine which could generate serious toxicity when administered for diabetic nephropathy.\u003c/p\u003e \u003cp\u003eIn terms of discovering the effective drugs that could overcome the side effects of the reference drugs mentioned above, the researchers have a broad interest in studying the biological activities of natural products since they are well-known to have withdrawal symptoms.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e Therefore, it is noteworthy to discover and further investigate the potency of secondary metabolites from natural products as new promising antidiabetic and anti-glycation agents.\u003c/p\u003e \u003cp\u003eFlavonoids, one of the secondary metabolites, are known to possess antidiabetic and anti-glycation properties.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e Due to their abundance in nature with known biological activities, flavones, flavonols, and flavanones can be highlighted among the several subclasses of flavonoids. Hence, it is exciting to explore the effectiveness of these constituents on antidiabetic and anti-glycation activities. Whereas, in this study, twelve selected natural flavonoids as portrayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e were examined.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMoreover, halogenated secondary metabolites have received increased attention in drug discovery and medicinal chemistry in recent decades. The insertion of halogen atoms into the moiety structures of natural products or synthetic compounds have known to affect biological activities.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e Halogens, especially lighter fluorine and chlorine, are widely used substituents in medicinal chemistry.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e In the case of bromine, many studies have been reported on the preparation of brominated flavonoids. In addition, a previous study reported that the brominated flavones exhibited better \u003cem\u003eα\u003c/em\u003e-glucosidase inhibition compared with their parent compounds and found that the number of bromine atoms on the B-ring affected the biological activity.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eHence, this study aims to investigate the effects of bromine atoms in the A-ring of the flavonoid scaffold on \u003cem\u003eα\u003c/em\u003e-glucosidase and \u003cem\u003eα\u003c/em\u003e-amylase inhibitory activities. Further investigation was carried out by performing the anti-glycation assay to examine whether those brominated flavonoids were also acting as glycation inhibitors which are further useful to treat diabetic complications. To date, this is the first report about the effects of those semisynthetic brominated flavonoids (flavones, flavonols, and flavanones) as antidiabetic and anti-glycation agents. All the selected natural flavonoids and their brominated compounds were examined on inhibitory activities against \u003cem\u003eα\u003c/em\u003e-glucosidase from \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e and \u003cem\u003eα\u003c/em\u003e-amylase from porcine pancreas. Moreover, since bovine serum albumin (BSA) could act as a model protein in the \u003cem\u003ein vitro\u003c/em\u003e experiment, hence the study also focused on the activity of those compounds toward BSA-glycation mediated MG.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eChemistry\u003c/h2\u003e \u003cp\u003eIn this study, four flavonoids including kaempferol (\u003cb\u003eF5\u003c/b\u003e), pinocembrin (\u003cb\u003eF8\u003c/b\u003e), pinostrobin (\u003cb\u003eF9\u003c/b\u003e), and alpinetin (\u003cb\u003eF10\u003c/b\u003e) were isolated from two plants material. The isolation of \u003cb\u003eF5\u003c/b\u003e was carried out by further purification of the MeOH extract of the flowers of \u003cem\u003eImpatiens balsamina\u003c/em\u003e L. using a silica gel column to yield \u003cb\u003eF5\u003c/b\u003e as a yellow powder \u003cb\u003e(\u003c/b\u003e69%). On another hand, three flavanones including \u003cb\u003eF8\u003c/b\u003e, \u003cb\u003eF9\u003c/b\u003e, and \u003cb\u003eF10\u003c/b\u003e were isolated from the MeOH extract of the rhizome of \u003cem\u003eBoesenbergia rotunda\u003c/em\u003e (L.) Mansf. The purification of selected fractions by silica gel column and recrystallization furnished a colorless crystal of \u003cb\u003eF8\u003c/b\u003e (46%), pale-yellow powder of \u003cb\u003eF9\u003c/b\u003e (16%), and \u003cb\u003eF10\u003c/b\u003e (8.1%). The structures of these isolated flavonoids were identified by NMR analysis and compared with previously reported data.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eSeveral protocols to introduce the bromine atom on the A-ring of the flavonoid scaffold were carried out. The synthesis of bromoflavonoids including bromoflavones, bromoflavonols, and bromoflavanones are depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo synthesize bromo derivatives of flavones (\u003cb\u003eF1a\u003c/b\u003e\u0026ndash;\u003cb\u003eF3a\u003c/b\u003e), a modified method described by Hairani and Chavasiri was carried out.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e The reaction was performed in the presence NaBr and oxone in acetone-H\u003csub\u003e2\u003c/sub\u003eO. The desired products \u003cb\u003eF1a\u003c/b\u003e, \u003cb\u003eF2a\u003c/b\u003e, and \u003cb\u003eF3a\u003c/b\u003e were characterized by the absence of signals of 6- and 8- protons. The NMR spectral data of \u003cb\u003eF1a\u003c/b\u003e, \u003cb\u003eF2a\u003c/b\u003e, and \u003cb\u003eF4a\u003c/b\u003e were also confirmed with those of the previous reports.\u003csup\u003e\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e From this series, \u003cb\u003eF3a\u003c/b\u003e is identified as a new semisynthetic compound. The selective bromination of baicalein at the C-8 position was carried out using a modified method described by Lee and coworkers using NBS in the presence of a catalytic amount of concentrated H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e at room temperature to yield \u003cb\u003eF4a\u003c/b\u003e and compared its NMR spectra with a previously reported data.\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e While, the preparation of \u003cb\u003eF5a\u003c/b\u003e and \u003cb\u003eF6a\u003c/b\u003e was performed by using NaBr and oxone in acetone-H\u003csub\u003e2\u003c/sub\u003eO, while, \u003cb\u003eF7a\u003c/b\u003e could be attained by using Br\u003csub\u003e2\u003c/sub\u003e in CH\u003csub\u003e3\u003c/sub\u003eCOOH.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e For bromoflavanones (\u003cb\u003eF8a\u003c/b\u003e\u0026ndash;\u003cb\u003eF12a\u003c/b\u003e) could also be obtained by using NaBr and oxone in acetone-H\u003csub\u003e2\u003c/sub\u003eO at room temperature for overnight. Among these series, \u003cb\u003eF10a\u003c/b\u003e was identified as a new semisynthetic compound.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBiological activity of natural flavonoids and their brominated compounds\u003c/h3\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003eIn vitro α-glucosidase inhibitory\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eAll selected flavonoids and their bromo derivatives were screened \u003cem\u003ein vitro\u003c/em\u003e for inhibitory \u003cem\u003eα\u003c/em\u003e-glucosidase activity which performed according to the previously described protocol.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e Acarbose was used as a positive control. The inhibition results at 50 \u003cem\u003e\u0026micro;\u003c/em\u003eM and IC\u003csub\u003e50\u003c/sub\u003e values for the compounds that exhibited\u0026thinsp;\u0026ge;\u0026thinsp;50% inhibition toward \u003cem\u003eα\u003c/em\u003e-glucosidase is described in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The criteria of IC\u003csub\u003e50\u003c/sub\u003e value is categorized as follow: IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;\u0026lt;\u0026thinsp;1 \u003cem\u003e\u0026micro;\u003c/em\u003eM (very strong), IC\u003csub\u003e50 =\u003c/sub\u003e 1\u0026ndash;10 \u003cem\u003e\u0026micro;\u003c/em\u003eM (strong), IC\u003csub\u003e50 =\u003c/sub\u003e 10.1\u0026ndash;30 \u003cem\u003e\u0026micro;\u003c/em\u003eM (relatively strong), IC\u003csub\u003e50 =\u003c/sub\u003e 30.1\u0026ndash;10 \u003cem\u003e\u0026micro;\u003c/em\u003eM (moderate), IC\u003csub\u003e50 =\u003c/sub\u003e 100.1\u0026ndash;200 \u003cem\u003e\u0026micro;\u003c/em\u003eM (weak), IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;\u0026gt;\u0026thinsp;200 \u003cem\u003e\u0026micro;\u003c/em\u003eM (not active).\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows various effects of bromine atoms on the inhibition of \u003cem\u003eα\u003c/em\u003e-glucosidase. The introduction of bulky groups such as bromine could be antagonistic or agonistic in comparison with the parent compound.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e In this screening study, the brominated compounds showed better activities compared with their parent ones. Nonetheless, some derivatives displayed no effects of bromine atoms or relatively have the same activity compared with the starting-material.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEnzymatic inhibitory activities of natural flavonoids and their brominated compounds.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCompound\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e\u003cem\u003e α\u003c/em\u003e-Glucosidase\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e\u003cem\u003eα\u003c/em\u003e-Amylase\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e% Inhibition\u003c/p\u003e \u003cp\u003eat 50 \u003cem\u003e\u0026micro;\u003c/em\u003eM*\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e (\u003cem\u003e\u0026micro;\u003c/em\u003eM)*\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e% Inhibition\u003c/p\u003e \u003cp\u003eat 50 \u003cem\u003e\u0026micro;\u003c/em\u003eM*\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e (\u003cem\u003e\u0026micro;\u003c/em\u003eM)*\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFlavones\u003c/b\u003e :\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChrysin (\u003cb\u003eF1\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e31.21\u0026thinsp;\u0026plusmn;\u0026thinsp;2.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6,8-Dibromochrysin (\u003cb\u003eF1a\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e89.70\u0026thinsp;\u0026plusmn;\u0026thinsp;1.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.68\u0026thinsp;\u0026plusmn;\u0026thinsp;3.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eApigenin (\u003cb\u003eF2\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e78.64\u0026thinsp;\u0026plusmn;\u0026thinsp;3.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e87.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e13.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.81\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6,8-Dibromoapigenin (\u003cb\u003eF2a\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e81.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e69.28\u0026thinsp;\u0026plusmn;\u0026thinsp;1.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLuteolin (\u003cb\u003eF3\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e34.37\u0026thinsp;\u0026plusmn;\u0026thinsp;2.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e97.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6,8-Dibromoluteolin (\u003cb\u003eF3a\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e95.08\u0026thinsp;\u0026plusmn;\u0026thinsp;2.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e98.57\u0026thinsp;\u0026plusmn;\u0026thinsp;1.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBaicalein (\u003cb\u003eF4\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e99.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e64.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8-Bromobaicalein (\u003cb\u003eF4a\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e99.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFlavonols\u003c/b\u003e :\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKaempferol (\u003cb\u003eF5\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e39.77\u0026thinsp;\u0026plusmn;\u0026thinsp;2.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e87.30\u0026thinsp;\u0026plusmn;\u0026thinsp;2.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6,8-Dibromokaempferol (\u003cb\u003eF5a\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e78.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e98.19\u0026thinsp;\u0026plusmn;\u0026thinsp;2.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMorin (\u003cb\u003eF6\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e97.47\u0026thinsp;\u0026plusmn;\u0026thinsp;1.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e51.82\u0026thinsp;\u0026plusmn;\u0026thinsp;4.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6,8-Dibromomorin (\u003cb\u003eF6a\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e99.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e98.65\u0026thinsp;\u0026plusmn;\u0026thinsp;1.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.69\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQuercetin (\u003cb\u003eF7\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e98.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e94.06\u0026thinsp;\u0026plusmn;\u0026thinsp;2.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6,8-Dibromoquercetin (\u003cb\u003eF7a\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e95.76\u0026thinsp;\u0026plusmn;\u0026thinsp;1.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e71.51\u0026thinsp;\u0026plusmn;\u0026thinsp;1.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e19.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFlavanones\u003c/b\u003e :\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePinocembrin (\u003cb\u003eF8\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25.23\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6,8-Dibromopinocembrin (\u003cb\u003eF8a\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e51.13\u0026thinsp;\u0026plusmn;\u0026thinsp;1.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePinostrobin (\u003cb\u003eF9\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15.13\u0026thinsp;\u0026plusmn;\u0026thinsp;3.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6,8-Dibromopinostrobin (\u003cb\u003eF9a\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e29.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20.82\u0026thinsp;\u0026plusmn;\u0026thinsp;4.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlpinetin (\u003cb\u003eF10\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e34.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6,8-Dibromoalpinetin (\u003cb\u003eF10a\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e43.82\u0026thinsp;\u0026plusmn;\u0026thinsp;1.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNaringenin (\u003cb\u003eF11\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e26.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e33.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6,8-Dibromonaringenin (\u003cb\u003eF11a\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e80.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e49.46\u0026thinsp;\u0026plusmn;\u0026thinsp;4.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHesperetin (\u003cb\u003eF12\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e45.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e29.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6,8-Dibromohesperetin (\u003cb\u003eF12a\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e66.26\u0026thinsp;\u0026plusmn;\u0026thinsp;2.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e40.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAcarbose\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e37.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e83.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e87.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e*Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;Standard deviation from two independent experiments and each performed in triplicate, n.d. = not determined\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eFor flavones subclasses, baicalein (\u003cb\u003eF4\u003c/b\u003e) which consists of three -OH groups on the A-ring exhibited very strong inhibition among flavones with a % inhibition of 99.42 and IC\u003csub\u003e50\u003c/sub\u003e value of 0.58 \u003cem\u003e\u0026micro;\u003c/em\u003eM. This result indicated that the number of the -OH groups on the A-ring of flavones might play a crucial role in the enzyme inhibition due to the fact that these could create more interactions with the enzyme \u003cem\u003evia\u003c/em\u003e hydrogen bonds and or electrostatic interactions.\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e Even though apigenin (\u003cb\u003eF2\u003c/b\u003e) also has three -OH groups, due to the position of one hydroxyl group on the B-ring, the inhibition of \u003cb\u003eF2\u003c/b\u003e was not as strong as baicalein (\u003cb\u003eF4\u003c/b\u003e). It showed relatively strong inhibition with a % inhibition of 78.64 and an IC\u003csub\u003e50\u003c/sub\u003e value of 22.74 \u003cem\u003e\u0026micro;\u003c/em\u003eM. This indicated that the -OH groups of flavones on the A-ring are more favorable for the interaction with the enzyme, whereas 5,6,7-trihydroxyflavone could be crucial substituents for enhancing \u003cem\u003eα\u003c/em\u003e-glucosidase inhibitory activity as stated by a previous study.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e For luteolin (\u003cb\u003eF3\u003c/b\u003e) containing four -OH groups exhibited only 34.37% inhibition. This could be concluded that the presence of the -OH group in the 3\u0026prime;-position of the B-ring slightly withdraws the activity of a compound.\u003c/p\u003e \u003cp\u003eComparing the natural flavones and their bromo derivatives, the results showed that the introduction of bromine atoms at positions 6- and 8- on chrysin (\u003cb\u003eF1a\u003c/b\u003e), apigenin (\u003cb\u003eF2a\u003c/b\u003e), and luteolin (\u003cb\u003eF3a\u003c/b\u003e) exhibited better activities compared with their parent compounds chrysin (\u003cb\u003eF1\u003c/b\u003e), apigenin (\u003cb\u003eF2\u003c/b\u003e), and luteolin (\u003cb\u003eF3\u003c/b\u003e) with % inhibition: 89.70, 81.98 and 95.08%, and IC\u003csub\u003e50\u003c/sub\u003e values: 2.97, 15.32 \u003cem\u003e\u0026micro;\u003c/em\u003eM, respectively. In contrast, the brominated baicalein (\u003cb\u003eF4a\u003c/b\u003e) showed a slight increase or no effect compared with its starting material. This could be indicated that the introduction of bromine atom at position 8- on flavones only give little effect or might not give an advantageous effect on \u003cem\u003eα\u003c/em\u003e-glucosidase inhibitory activity.\u003c/p\u003e \u003cp\u003eFor flavonols, the results showed that the natural flavonols including morin (\u003cb\u003eF6\u003c/b\u003e) and quercetin (\u003cb\u003eF7\u003c/b\u003e) containing five -OH groups displayed better activities than kaempferol (\u003cb\u003eF5\u003c/b\u003e) which only four -OH groups. Even though morin (\u003cb\u003eF6\u003c/b\u003e) and quercetin (\u003cb\u003eF7\u003c/b\u003e) have the same number of -OH groups, the different positions of the -OH group lead to the different power of compounds to reduce \u003cem\u003eα\u003c/em\u003e-glucosidase activity, whereas quercetin (\u003cb\u003eF7\u003c/b\u003e) showed strong inhibition with an IC\u003csub\u003e50\u003c/sub\u003e value 1.33 \u003cem\u003e\u0026micro;\u003c/em\u003eM. This implied that the catechol moiety (\u003cem\u003eortho\u003c/em\u003e -OH groups) in \u003cb\u003eF7\u003c/b\u003e gave the benefit towards the \u003cem\u003eα\u003c/em\u003e-glucosidase inhibitory activity over the \u003cem\u003emeta\u003c/em\u003e -OH groups.\u003c/p\u003e \u003cp\u003eMoreover, for the brominated flavonols, \u003cb\u003eF5a\u003c/b\u003e and \u003cb\u003eF6a\u003c/b\u003e displayed relatively strong inhibition and were better than their parent compounds with IC\u003csub\u003e50\u003c/sub\u003e values: 13.89 and 8.28 \u003cem\u003e\u0026micro;\u003c/em\u003eM, respectively. In contrast, brominated quercetin (\u003cb\u003eF7a\u003c/b\u003e) did not give a crucial effect on \u003cem\u003eα\u003c/em\u003e-glucosidase activity whereas the activity slightly declines. This behavior might be due to the bulky compounds compared with the starting material which is then difficult to reach the binding site of the enzyme. However, \u003cb\u003eF7a\u003c/b\u003e (IC\u003csub\u003e50\u003c/sub\u003e value 3.90 \u003cem\u003e\u0026micro;\u003c/em\u003eM) showed a better effect compared with \u003cb\u003eF5a\u003c/b\u003e and \u003cb\u003eF6a\u003c/b\u003e. In some cases, the hydrophobicity of the compound played an important effect on enzyme activity which led to an increase in the hydrophobic interaction between the compound and enzyme. The value of log P (octanol-water partition coefficient) could be used to describe how hydrophilic or hydrophobic a compound. Comparing the \u003csup\u003ec\u003c/sup\u003eLog P values of \u003cb\u003eF6a\u003c/b\u003e (1.331) and \u003cb\u003eF7a (\u003c/b\u003e1.701) whereas \u003cb\u003eF7a\u003c/b\u003e was more hydrophobic than \u003cb\u003eF6a\u003c/b\u003e, therefore \u003cb\u003eF7a\u003c/b\u003e was stronger than \u003cb\u003eF6a\u003c/b\u003e due to the possibility of \u003cb\u003eF7a\u003c/b\u003e having more hydrophobic interactions with the enzyme.\u003c/p\u003e \u003cp\u003eMost of the natural flavanones and their brominated compounds showed weak inhibition, except for compounds \u003cb\u003eF11a\u003c/b\u003e and \u003cb\u003eF12a\u003c/b\u003e with relatively strong inhibition. This phenomenon indicated that the absence of a double bond at C-2 and C-3 positions led to the lessening of the \u003cem\u003eα\u003c/em\u003e-glucosidase inhibitory activity. Compound \u003cb\u003eF11a\u003c/b\u003e (IC\u003csub\u003e50\u003c/sub\u003e 13.63 \u003cem\u003e\u0026micro;\u003c/em\u003eM) showed better inhibition compared with \u003cb\u003eF12a\u003c/b\u003e (IC\u003csub\u003e50\u003c/sub\u003e 16.70 \u003cem\u003e\u0026micro;\u003c/em\u003eM) due to the -OH group at 4\u0026prime;-position being replaced by a -OMe group leading to a slight decrease the activity.\u003c/p\u003e \u003cp\u003eAccording to the findings, the structure-activity relationship of natural and brominated flavonoids could be rationalized into several important points as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, Lineweaver-Burk plot was established to illustrate the kinetic inhibition of \u003cem\u003eα\u003c/em\u003e-glucosidase in the presence of \u003cb\u003eF4a\u003c/b\u003e. The plot of 1/v \u003cem\u003evs\u003c/em\u003e 1/[p-NPG] for \u003cb\u003eF4a\u003c/b\u003e, gave straight lines that intersected at the same point in the second quadrant, indicating that \u003cb\u003eF4a\u003c/b\u003e was a mixed-type inhibitor (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). This result indicated that \u003cb\u003eF4a\u003c/b\u003e could bind with free enzymes, and enzyme-substrate complex to reduce the catalytic activity of \u003cem\u003eα\u003c/em\u003e-glucosidase.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition, the equilibrium constants of binding of \u003cb\u003eF4a\u003c/b\u003e to free enzyme (\u003cem\u003eK\u003c/em\u003e\u003csub\u003ei\u003c/sub\u003e) and enzyme-substrate complex (\u003cem\u003eK\u003c/em\u003e\u003csub\u003eis\u003c/sub\u003e) through plots of slope (\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e\u003cem\u003e/V\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e) and vertical intercept (1\u003cem\u003e/V\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e) \u003cem\u003evs\u003c/em\u003e \u003cb\u003eF4a\u003c/b\u003e with various concentrations were also determined. From the calculation, the \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e value was 1.37 \u003cem\u003e\u0026micro;\u003c/em\u003eM while \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eis\u003c/em\u003e\u003c/sub\u003e value was 2.53 \u003cem\u003e\u0026micro;\u003c/em\u003eM. The \u003cem\u003eK\u003c/em\u003e\u003csub\u003eis\u003c/sub\u003e value of \u003cb\u003eF4a\u003c/b\u003e was higher than its \u003cem\u003eK\u003c/em\u003e\u003csub\u003ei\u003c/sub\u003e value, suggesting that the affinity of \u003cb\u003eF4a\u003c/b\u003e with free enzyme was higher than that with the enzyme-substrate complex.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIn vitro α-amylase inhibitory activity\u003c/h3\u003e\n\u003cp\u003eThe \u003cem\u003eα\u003c/em\u003e-amylase assay was performed in 96-well microplates using a final volume of 200 \u003cem\u003e\u0026micro;\u003c/em\u003eL as the previous method with some modification.\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e The screening at 50 \u003cem\u003e\u0026micro;\u003c/em\u003eM (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) showed that flavone and flavonol sub-classes demonstrated better inhibition against \u003cem\u003eα\u003c/em\u003e-amylase than those of flavanones. This could be due to the presence of a double bond at positions C-2 and C-3 in flavones and flavonols that might give an inhibition effect on the enzyme. In contrast with flavanones, this sub-class showed relatively lower inhibition. Subsequently, the tested compounds that showed\u0026thinsp;\u0026ge;\u0026thinsp;70% inhibition on \u003cem\u003eα\u003c/em\u003e-amylase was further determined for their IC\u003csub\u003e50\u003c/sub\u003e values.\u003c/p\u003e \u003cp\u003eAmong natural flavones, increasing the number of -OH groups could increase the inhibition level. Chrysin (\u003cb\u003eF1\u003c/b\u003e) with two -OH groups on the A-ring only showed 22.59% inhibition. Nevertheless, the activity of the compound with another additional -OH group on the A-ring (\u003cb\u003eF4\u003c/b\u003e) was four-fold better than \u003cb\u003eF1\u003c/b\u003e. Even better activity could be reached when the compound contained the additional group on the B-ring, hence for compounds \u003cb\u003eF2\u003c/b\u003e (one additional -OH) and \u003cb\u003eF3\u003c/b\u003e (two additional -OH groups) exhibited IC\u003csub\u003e50\u003c/sub\u003e values of 13.18 and 2.18 \u003cem\u003e\u0026micro;\u003c/em\u003eM, respectively. This indicated that the -OH groups could be one of the factors that could increase the interaction number between the compound and enzyme leading to higher inhibition. The same phenomenon was also observed for natural flavonols, kaempferol (\u003cb\u003eF5\u003c/b\u003e) consisting of four -OH groups showed an IC\u003csub\u003e50\u003c/sub\u003e value of 10.43 \u003cem\u003e\u0026micro;\u003c/em\u003eM followed by quercetin (\u003cb\u003eF7\u003c/b\u003e) with five -OH groups displayed higher inhibition (IC\u003csub\u003e50\u003c/sub\u003e value 11.16 \u003cem\u003e\u0026micro;\u003c/em\u003eM). In contrast, the opposite effect appeared for morin (\u003cb\u003eF6\u003c/b\u003e), due to the different position of -OH groups (\u003cem\u003emeta\u003c/em\u003e), the activity was weaker than in \u003cb\u003eF7\u003c/b\u003e. Furthermore, the results also displayed that luteolin (\u003cb\u003eF3\u003c/b\u003e) was better than quercetin (\u003cb\u003eF7\u003c/b\u003e) which indicated that the presence of the -OH group at 3-position on the C-ring was not essential for \u003cem\u003eα\u003c/em\u003e-amylase inhibitory activity. This result was in line with a previous study which indicated that -OH groups at 4\u0026prime;- and 5\u0026prime;-position increased the inhibitory activity against \u003cem\u003eα\u003c/em\u003e-amylase.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e While natural flavanones and their brominated flavanones were found to be weak and not active agents toward \u003cem\u003eα\u003c/em\u003e-amylase inhibitory activity whereas the percent inhibition was in the range of 12.85 to 51.13%.\u003c/p\u003e \u003cp\u003eThe introduction of bromine atoms into the flavonoid scaffold provided varied results. Among these tested compounds, \u003cb\u003eF3a\u003c/b\u003e displayed the highest inhibition with an IC\u003csub\u003e50\u003c/sub\u003e value of 0.99 \u003cem\u003e\u0026micro;\u003c/em\u003eM, which was six-fold better than the reference drug acarbose (IC\u003csub\u003e50\u003c/sub\u003e value of 6.73 \u003cem\u003e\u0026micro;\u003c/em\u003eM).\u003c/p\u003e \u003cp\u003eAccording to the findings, the structure-activity relationship of natural and brominated flavonoids could be rationalized into several important points as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe Lineweaver-Burk plot was also established to illustrate the kinetic inhibition of \u003cem\u003eα\u003c/em\u003e-amylase in the presence of \u003cb\u003eF3a\u003c/b\u003e. The plot of 1/v \u003cem\u003evs\u003c/em\u003e 1/[CNP-G3] for \u003cb\u003eF3a\u003c/b\u003e, showed that the straight lines intersected at the same point in the second quadrant, which specified that \u003cb\u003eF3a\u003c/b\u003e was a mixed-type inhibitor (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e) in which \u003cb\u003eF3a\u003c/b\u003e could bind with free enzymes, as well as enzyme-substrate complex to reduce the catalytic activity of \u003cem\u003eα\u003c/em\u003e-amylase.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMoreover, the equilibrium constants of binding of \u003cb\u003eF3a\u003c/b\u003e to free enzyme (\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e) and enzyme-substrate complex (\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eis\u003c/em\u003e\u003c/sub\u003e) through plots of slope (\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e\u003cem\u003e/V\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e) and vertical intercept (1\u003cem\u003e/V\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e) \u003cem\u003evs\u003c/em\u003e \u003cb\u003eF3a\u003c/b\u003e with various concentrations were also determined. The value of \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e was 0.18 \u003cem\u003e\u0026micro;\u003c/em\u003eM while \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eis\u003c/em\u003e\u003c/sub\u003e values was 0.37 \u003cem\u003e\u0026micro;\u003c/em\u003eM suggesting that the affinity of \u003cb\u003eF3a\u003c/b\u003e with free enzyme was higher than that with the enzyme-substrate complex.\u003c/p\u003e\n\u003ch3\u003eIn vitro BSA-glycation mediated methyglyoxal (MG) inhibitory activity\u003c/h3\u003e\n\u003cp\u003eIncreased glucose level is known as the typical indicator of DM, in which glucose as a reducing sugar is conducted to non-enzymatic glycation of many proteins including plasma protein, especially albumin.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e Bovine serum albumin (BSA) was the glycation induced by a high reactive dicarbonyl species like methylglyoxal (MG), which reacted with proteins to form irreversible heterogeneous-AGEs. Since BSA may serve as a model protein for \u003cem\u003ein vitro\u003c/em\u003e experiments, therefore in this study, the effect of tested compounds was applied to the \u003cem\u003ein vitro\u003c/em\u003e BSA-glycation mediated by MG.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eInhibitory activity of natural flavonoids and the brominated flavonoids toward BSA-glycation mediated methylglyoxal (MG).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCompound\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e% Inhibition at 500 \u003cem\u003e\u0026micro;\u003c/em\u003eM*\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e (\u003cem\u003e\u0026micro;\u003c/em\u003eM)*\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFlavones\u003c/b\u003e :\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChrysin (\u003cb\u003eF1\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e53.96\u0026thinsp;\u0026plusmn;\u0026thinsp;2.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6,8-Dibromochrysin (\u003cb\u003eF1a\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e97.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eApigenin (\u003cb\u003eF2\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e73.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6,8-Dibromoapigenin (\u003cb\u003eF2a\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e97.81\u0026thinsp;\u0026plusmn;\u0026thinsp;2.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e57.01\u0026thinsp;\u0026plusmn;\u0026thinsp;5.37\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLuteolin (\u003cb\u003eF3\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e96.32\u0026thinsp;\u0026plusmn;\u0026thinsp;1.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e85.63\u0026thinsp;\u0026plusmn;\u0026thinsp;4.97\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6,8-Dibromoluteolin (\u003cb\u003eF3a\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e95.37\u0026thinsp;\u0026plusmn;\u0026thinsp;1.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e79.48\u0026thinsp;\u0026plusmn;\u0026thinsp;1.10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBaicalein (\u003cb\u003eF4\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e75.66\u0026thinsp;\u0026plusmn;\u0026thinsp;1.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8-Bromobaicalein (\u003cb\u003eF4a\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e36.68\u0026thinsp;\u0026plusmn;\u0026thinsp;1.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFlavonols\u003c/b\u003e :\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKaempferol (\u003cb\u003eF5\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e76.47\u0026thinsp;\u0026plusmn;\u0026thinsp;1.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6,8-Dibromokaempferol (\u003cb\u003eF5a\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e72.24\u0026thinsp;\u0026plusmn;\u0026thinsp;1.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMorin (\u003cb\u003eF6\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e94.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e132.45\u0026thinsp;\u0026plusmn;\u0026thinsp;2.47\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6,8-Dibromomorin (\u003cb\u003eF6a\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e92.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e61.22\u0026thinsp;\u0026plusmn;\u0026thinsp;5.98\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQuercetin (\u003cb\u003eF7\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100,00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e68.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.74\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6,8-Dibromoquercetin (\u003cb\u003eF7a\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e60.54\u0026thinsp;\u0026plusmn;\u0026thinsp;2.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFlavanones\u003c/b\u003e :\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePinocembrin (\u003cb\u003eF8\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e47.60\u0026thinsp;\u0026plusmn;\u0026thinsp;3.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6,8-Dibromopinocembrin (\u003cb\u003eF8a\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e46.31\u0026thinsp;\u0026plusmn;\u0026thinsp;2.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePinostrobin (\u003cb\u003eF9\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.90\u0026thinsp;\u0026plusmn;\u0026thinsp;3.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6,8-Dibromopinostrobin (\u003cb\u003eF9a\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e37.21\u0026thinsp;\u0026plusmn;\u0026thinsp;7.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlpinetin (\u003cb\u003eF10\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e45.65\u0026thinsp;\u0026plusmn;\u0026thinsp;2.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6,8-Dibromoalpinetin (\u003cb\u003eF10a\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e56.89\u0026thinsp;\u0026plusmn;\u0026thinsp;4.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNaringenin (\u003cb\u003eF11\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e83.98\u0026thinsp;\u0026plusmn;\u0026thinsp;3.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6,8-Dibromonaringenin (\u003cb\u003eF11a\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e51.62\u0026thinsp;\u0026plusmn;\u0026thinsp;2.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e64.51\u0026thinsp;\u0026plusmn;\u0026thinsp;6.87\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHesperetin (\u003cb\u003eF12\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e51.62\u0026thinsp;\u0026plusmn;\u0026thinsp;5.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6,8-Dibromohesperetin (\u003cb\u003eF12a\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e63.27\u0026thinsp;\u0026plusmn;\u0026thinsp;2.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAminoguanidine hydrochloride\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e79.50\u0026thinsp;\u0026plusmn;\u0026thinsp;1.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e240.05\u0026thinsp;\u0026plusmn;\u0026thinsp;5.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e*Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;Standard deviation from two independent experiments and each performed in triplicate.\u003c/p\u003e \u003cp\u003eAll selected flavonoids (\u003cb\u003eF1\u003c/b\u003e\u0026ndash;\u003cb\u003eF12\u003c/b\u003e) and their brominated compounds (\u003cb\u003eF1a\u003c/b\u003e\u0026ndash;\u003cb\u003eF12a\u003c/b\u003e) were evaluated for their anti-glycation potential. From the screening result at 500 \u0026micro;M of tested compounds in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, several flavonoids and brominated flavonoids significantly inhibited BSA glycation mediated by MG which showed better inhibition (93.40 to 99.75%) compared with its positive control, aminoguanidine hydrochloride (79.66%). Flavones and their brominated gave the inhibition in the range of 35.56\u0026ndash;97.82%, while the inhibition ranging from 60.85 to 99.75% was shown by flavonols and their brominated derivatives. In addition, flavanones and their brominated compounds displayed inhibition in the range of 11.90\u0026ndash;96.53%.\u003c/p\u003e \u003cp\u003eAmong natural flavones, the increasing percent inhibition was observed by increasing the number -of OH groups with the order: chrysin (\u003cb\u003eF1\u003c/b\u003e)\u0026thinsp;\u0026lt;\u0026thinsp;apigenin (\u003cb\u003eF2\u003c/b\u003e)\u0026thinsp;\u0026lt;\u0026thinsp;baicalein (\u003cb\u003eF4\u003c/b\u003e)\u0026thinsp;\u0026lt;\u0026thinsp;luteolin (\u003cb\u003eF3\u003c/b\u003e). This phenomenon indicated that the -OH groups played a crucial role in inhibiting the glycation process because they increased the number of intermolecular hydrogen bonds whereas the lower energy barriers were accompanied by more H-bonds.\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e Moreover, the C\u0026thinsp;=\u0026thinsp;O group of the C-ring facilitated the intermolecular H-bond formation, in which the flavonoid could effectively hinder the AGEs formation.\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e In addition, previously, it has been reported that the -OH group at the 5-position on the A-ring was essential for trapping MG by flavonoids.\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eFurthermore, two brominated flavones (\u003cb\u003eF1a\u003c/b\u003e and \u003cb\u003eF2a\u003c/b\u003e) exhibited better activities than their parent compounds (\u003cb\u003eF1\u003c/b\u003e and \u003cb\u003eF2\u003c/b\u003e). This implied that the presence of bromine atoms on the A-ring gave the advantage effect for the activity due to the fact that halogens could act as hydrogen bond acceptors and potentially make a more favorable contribution to the binding. In contrast, the observed \u003cb\u003eF4a\u003c/b\u003e containing only one bromine atom on the A-ring at 8-position exhibited lower inhibition compared with the starting material (\u003cb\u003eF4\u003c/b\u003e). This described that the presence of bromine atom at 8-position did not give a beneficial effect on the activity. The introduction of bromine atom at 6-position on the A-ring of flavone could enhance the inhibition of the glycation process. However, this outcome only occurred for flavone containing two -OH groups on the A-ring and one additional -OH group on the B-ring. For example, 6,8-dibromoluteolin (\u003cb\u003eF3a\u003c/b\u003e) with two more additional -OH groups on the B-ring displayed no increased effect compared with its parent compound luteolin (\u003cb\u003eF3\u003c/b\u003e). This specified that the inhibition was favored when the inhibitor was not a bulky compound that may offer steric hindrance.\u003c/p\u003e \u003cp\u003eIn the case of natural flavonols, the same trend of the number -OH groups were also observed which the percent inhibition of glycation follows the order: kaempferol (\u003cb\u003eF5\u003c/b\u003e)\u0026thinsp;\u0026lt;\u0026thinsp;morin (\u003cb\u003eF6\u003c/b\u003e)\u0026thinsp;\u0026lt;\u0026thinsp;quercetin (\u003cb\u003eF7\u003c/b\u003e). Albeit \u003cb\u003eF6\u003c/b\u003e and \u003cb\u003eF7\u003c/b\u003e contained the same number of -OH groups, due to the different positions, the activity showed a slight dissimilar effect. This denoted that the catechol moiety on the B-ring was favored over the \u003cem\u003emeta\u003c/em\u003e-OH. In addition, the introduction of bromine atoms at 6- and 8- positions of flavonols (\u003cb\u003eF5a\u003c/b\u003e\u0026ndash;\u003cb\u003eF7a\u003c/b\u003e) exhibited a decrease in inhibition compared with their starting materials. This due to the bulky compounds were generated from this series of derivatives leading to the difficulty to access the inhibition.\u003c/p\u003e \u003cp\u003eFor flavanones, the importance of the -OH group at the 5-position of flavonoids was also observed when investigated pinocembrin (\u003cb\u003eF8\u003c/b\u003e) containing two -OH group at 5,7-positions and pinostrobin (\u003cb\u003eF9\u003c/b\u003e) with one -OH group at 7-position and one -OMe group at 5-position, displayed a significant difference in the inhibition. The same result was also observed when the 7-OH was replaced by 7-OMe which was possessed by alpinetin (\u003cb\u003eF10\u003c/b\u003e), which revealed that the -OH group at the 7-position played a crucial role in inhibiting the glycation process. Furthermore, the increase of the number -OH groups in flavanones was also essential for the activity, for instance, pinocembrin (\u003cb\u003eF8\u003c/b\u003e) contained two -OH groups on the A-ring with inhibition of 47.60%, then underwent slightly increase inhibition for naringenin (\u003cb\u003eF11\u003c/b\u003e) which contained one more additional -OH group on the B-ring with inhibition of 56.89%. But then slightly reduce the inhibition level once the -OH group at 4-position in the B-ring was replaced by -OMe, even though compound \u003cb\u003eF12\u003c/b\u003e contained one more -OH group at 3\u0026prime;-position on the B-ring. This revealed that the -OH group at the 4\u0026prime;-position in the B-ring of flavanone was favored over the 3\u0026prime;-position. As stated in a previous study that the presence of one -OH and one -OMe group showed varied activity depending upon the position of the -OH substituent, whereas a compound with an \u003cem\u003eortho\u003c/em\u003e hydroxy was found to be a not good inhibitor.\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eSubsequently, several selected compounds that exhibited percent inhibition\u0026thinsp;\u0026ge;\u0026thinsp;80% were further investigated for their IC\u003csub\u003e50\u003c/sub\u003e values by varying the concentrations as depicted in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The criteria of IC\u003csub\u003e50\u003c/sub\u003e value is categorized as follow: IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;\u0026lt;\u0026thinsp;10 \u003cem\u003e\u0026micro;\u003c/em\u003eM (very strong), IC\u003csub\u003e50 =\u003c/sub\u003e 10.1\u0026ndash;100 \u003cem\u003e\u0026micro;\u003c/em\u003eM (strong), IC\u003csub\u003e50 =\u003c/sub\u003e 100.1\u0026ndash;200 \u003cem\u003e\u0026micro;\u003c/em\u003eM (relatively strong), IC\u003csub\u003e50 =\u003c/sub\u003e 200.1\u0026ndash;300 \u003cem\u003e\u0026micro;\u003c/em\u003eM (moderate), IC\u003csub\u003e50 =\u003c/sub\u003e 300.1\u0026ndash;500 \u003cem\u003e\u0026micro;\u003c/em\u003eM (weak), IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;\u0026gt;\u0026thinsp;500 \u003cem\u003e\u0026micro;\u003c/em\u003eM (not active).\u003c/p\u003e \u003cp\u003eAccording to the results, 6,8-dibromochrysin (\u003cb\u003eF1a\u003c/b\u003e) showed the highest inhibition with an IC\u003csub\u003e50\u003c/sub\u003e value of 50.90 \u003cem\u003e\u0026micro;\u003c/em\u003eM, followed by compounds \u003cb\u003eF2a\u003c/b\u003e (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;57.01 \u003cem\u003e\u0026micro;\u003c/em\u003eM), \u003cb\u003eF6a\u003c/b\u003e (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;61.22 \u0026micro;M), \u003cb\u003eF11a\u003c/b\u003e (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;64.51 \u003cem\u003e\u0026micro;\u003c/em\u003eM), \u003cb\u003eF7\u003c/b\u003e (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;68.47 \u003cem\u003e\u0026micro;\u003c/em\u003eM), \u003cb\u003eF3a\u003c/b\u003e (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;79.48 \u003cem\u003e\u0026micro;\u003c/em\u003eM), and \u003cb\u003eF3\u003c/b\u003e (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;85.6 \u003cem\u003e\u0026micro;\u003c/em\u003eM), in which these compounds displayed strong inhibition toward the BSA-glycation process mediated MG. It seems that the anti-glycation activity favored the compound that less bulky. Moreover, morin (\u003cb\u003eF6\u003c/b\u003e) showed a relatively strong effect as an anti-glycation agent with an IC\u003csub\u003e50\u003c/sub\u003e value of 132.45 \u003cem\u003e\u0026micro;\u003c/em\u003eM. All these selected compounds mainly showed 2- to 6-fold better activities compared with its standard drug, aminoguanidine hydrochloride (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;240.05 \u003cem\u003e\u0026micro;\u003c/em\u003eM).\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003ePlant Materials\u003c/h2\u003e \u003cp\u003eTwo plant materials were used in this work including the rhizomes of \u003cem\u003eBoesenbergia rotunda\u003c/em\u003e (L.) Mansf. which was purchased from the herbal drug store in Bangkok-Thailand, and the flowers of \u003cem\u003eImpatiens balsamina\u003c/em\u003e L. was collected from Indonesia.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eEquipment and Instruments\u003c/h2\u003e \u003cp\u003e \u003csup\u003e \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e \u003c/sup\u003eH NMR (500 MHz) and \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR (125 MHz) spectra were recorded with a JEOL spectrometer (JNM-ECZ500R/S1) while \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR (400 MHz) and \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR (100 MHz) spectra were recorded on a Bruker 400 AVANCE spectrometer, and chemical shifts were recorded in parts per million (ppm) and coupling constants (\u003cem\u003eJ\u003c/em\u003e) were given in Hertz. The LC-QTOF-MS/MS analysis was performed on an Agilent HPLC 1260 series coupled with a QTOF 6540 UHD accurate mass (Agilent Technologies, Waldbronn, Germany). The fluorescence intensity was measured by the EnSight Multimode Plate Reader PerkinElmer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eChemicals\u003c/h2\u003e \u003cp\u003eChrysin, apigenin, luteolin, baicalein, quercetin hydrate, naringenin, and hesperetin were purchased from Tokyo Chemical Industry company, while morin was bought from Fluka and used without further purification. Other synthetic reagents were purchased from Sigma-Aldrich company or otherwise stated. All solvents used in this study were purified by standard methods, except for those which were reagent grades. The progress of the reaction was monitored by Thin Layer Chromatography (TLC) using TLC silica gel 60 F\u003csub\u003e254\u003c/sub\u003e Merck. Purification of semisynthetic compounds was performed by column chromatography using silica gel (70\u0026ndash;230 mesh) of SiliaFlash\u0026reg; G60 (Canada). \u003cem\u003eα\u003c/em\u003e-Glucosidase (Sigma G5003) derived from Baker\u0026rsquo;s yeast, \u003cem\u003eα\u003c/em\u003e-amylase from porcine pancreas type VI-B, 4-nitrophenyl \u003cem\u003eα\u003c/em\u003e-D-glucopyranoside (\u003cem\u003ep-\u003c/em\u003eNPG, Sigma N1377), 2-chloro-4-nitrophenyl \u003cem\u003eα\u003c/em\u003e-D-maltotrioside (CNP-G3), methylglyoxal solution, acarbose, and bovine serum albumin (BSA) were purchased from Sigma-Aldrich. Aminoguanidine hydrochloride was used as a positive control for anti-glycation and was purchased from Tokyo Chemical Industry company.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMethods\u003c/h2\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003eIsolation of kaempferol (F5) from Impatiens balsamina L. flowers\u003c/h2\u003e \u003cp\u003eThe dried and powdered \u003cem\u003eImpatiens balsamina\u003c/em\u003e L. flowers (272 g) was done by maceration in MeOH for three days at room temperature, filtered, and concentrated by a rotary evaporator. This step was repeated three times to obtain the dark brown MeOH crude extract (97.62 g, 36%). This crude extract was then partitioned with EtOAc and H\u003csub\u003e2\u003c/sub\u003eO and separated. EtOAc fraction was then concentrated to obtain 38.82 g of EtOAc extract. Subsequently, this fraction was subjected to silica gel column which was initially eluted with hexane-EtOAc and EtOAc-MeOH by increasing polarity to give five fractions (1\u0026ndash;5). The precipitate from the fraction 1 was then collected and washed with hexane:EtOAc (1:1) to give kaempferol (\u003cb\u003eF5\u003c/b\u003e) as yellow powder (92 mg). Fraction 2 (1.46 g) was then further purified by using silica gel column and eluted with hexane-EtOAc to give six subfractions (2a\u0026ndash;2f). Subfractions 2d and 2e formed precipitates. Washing the precipitate using hexane:EtOAc (1:1) gave 1.0 g of kaempferol (\u003cb\u003eF5\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eKaempferol (\u003cb\u003eF5\u003c/b\u003e): yellow powder (0.4%), \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ (ppm) 12.47 (s, 5-OH), 10.80 (s, 3-OH), 10.12 (s, 7-OH), 9.37 (s, 4\u0026prime;-OH), 8.04 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8 Hz, 2H), 6.92 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.8 Hz, 2H), and 6.19 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.4 Hz, 1H); \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ (ppm) 175.9, 163.9, 160.7, 159.2, 156.2, 146.9, 135.7, 129.5, 121.7, 115.5, 103.1, 98.2, and 93.5.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003eIsolation of pinocembrin (F8), pinostrobin (F9) and alpinetin (F10) from Boesenbergia rotunda (L.) Mansf. rhizomes\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eThe extraction of the dried and powdered \u003cem\u003eBoesenbergia rotunda\u003c/em\u003e (L.) Mansf. rhizomes (5.0 kg) was done by maceration in MeOH for three days at room temperature, filtered, and concentrated by a rotary evaporator. This step was repeated three times to obtain the dark brown MeOH crude extract (700 g, 14%). This crude extract was then subjected to silica gel column which was initially eluted with hexane-EtOAc and EtOAc-MeOH by increasing polarity to give seven fractions. Washing the precipitate in fraction 5 using EtOAc gave pinocembrin (\u003cb\u003eF8\u003c/b\u003e) as pale-yellow solid 113 g (16%). After recrystallization of precipitates from fractions 2, 3, and 4 with hexane and EtOAc, pinostrobin (\u003cb\u003eF9\u003c/b\u003e) as colorless crystal 320 g (46%) was attained. In addition, recrystallization of the precipitate in fraction 6 using CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e and MeOH, furnished alpinetin (\u003cb\u003eF10\u003c/b\u003e) as a pale-yellow solid 57 g (8.1%).\u003c/p\u003e \u003cp\u003ePinocembrin (\u003cb\u003eF8\u003c/b\u003e): pale-yellow solid (16%), \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR (500 MHz, Acetone-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ (ppm) 12.16 (s, 5-OH), 9.76 (s, 7-OH), 7.57 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.0, 1.5 Hz, 2H), 7.42 (m, 3H), 6.00 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.5 Hz, 1H), 5.97 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.0 Hz, 1H), 5.57 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;13.0, 3.5 Hz, 1H), 3.17 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;17.0, 12.5 Hz, 1H), and 2.81 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;17.0, 3.0 Hz, 1H); \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR (125 MHz, Acetone-\u003cem\u003ed6\u003c/em\u003e) δ (ppm) 196.8, 167.4, 165.2, 164.1, 140.0, 129.4, 127.3, 103.2, 96.9, 95.9, 79.9, and 43.5.\u003c/p\u003e \u003cp\u003ePinostrobin (\u003cb\u003eF9\u003c/b\u003e): colorless crystal (46%), \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR (500 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) δ (ppm) 12.02 (s, 5-OH), 7.42 (m, 5H), 6.08 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.5 Hz, 1H), 6.07 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.0 Hz, 1H), 5.42 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;13.5, 3.5 Hz, 1H), 3.81 (s, 3H), 3.09 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;17.0, 13.0 Hz, 1H), and 2.83 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;17.0, 3.0 Hz, 1H); \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR (125 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) δ (ppm) 196.3, 168.5, 164.3, 162.9, 138.5, 129.0, 126.3, 103.3, 95.3, 94.4, 79.4, 55.8, and 43.5.\u003c/p\u003e \u003cp\u003eAlpinetin (\u003cb\u003eF10\u003c/b\u003e): pale-yellow solid (8.1%), \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR (500 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ (ppm) 10.57 (s, 7-OH), 7.49 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.5, 1.5 Hz, 2H), 7.39 (m, 3H), 6.04 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;35.0, 2.0 Hz, 1H), 5.48 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;12.5, 3.0 Hz, 1H), 3.74 (s, 3H), 2.98 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;16.5, 12.5 Hz, 1H), and 2.62 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;16.5, 3.5 Hz, 1H); \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR (125 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ (ppm) 187.4, 164.4, 164.1, 162.2, 139.2, 128.5, 128.3, 126.5, 104.5, 95.7, 93.4. 78.1, 55.6, and 44.9.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eSynthesis of bromoflavones\u003c/h2\u003e \u003cp\u003eBromination of chrysin, apigenin and luteolin was performed according to the previously method.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e 6,8-Dibromochrysin (\u003cb\u003eF1a\u003c/b\u003e) was obtained by reacting Chrysin (\u003cb\u003eF1\u003c/b\u003e,1 mmol) in acetone:water 5:1 and NaBr (3 mmol). After cooling, oxone (3 mmol) was added and the mixture was stirred at room temperature for 3 h. The final solution was treated with Na\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e and evaporated under reduced pressure. The residue was recrystallized from MeOH to yield 92% yellow powder as a brominated product (\u003cb\u003eF1a\u003c/b\u003e). The same procedure was applied for synthesizing 6,8-dibromoapigenin (\u003cb\u003eF2a\u003c/b\u003e) and 6,8-dibromoluteolin (\u003cb\u003eF3a\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eWhile the brominated baicalein (\u003cb\u003eF4a\u003c/b\u003e) was obtained by mixing 1 mmol baicalein (F4) and 1 mmol \u003cem\u003eN\u003c/em\u003e-bromosuccinimide (NBS) in 4.0 mL tetrahydrofuran (THF) in the presence of 5.0 \u003cem\u003e\u0026micro;\u003c/em\u003eL concentrated H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e as described by a previously method.\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e The reaction mixture was stirred at room temperature for 12 h before extraction with EtOAc. The precipitated product was washed with 10% aqueous NaHSO\u003csub\u003e4\u003c/sub\u003e solution, dried over anhydrous Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, and concentrated under reduced pressure. The residue was recrystallized from MeOH to the target compound (58%) as a yellow powder.\u003c/p\u003e \u003cp\u003e6,8-Dibromochrysin (\u003cb\u003eF1a\u003c/b\u003e): yellow powder (92% yield). \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR (500 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ (ppm) 8.10 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.5 Hz, 2H), 7.60 (m, 3H), and 7.14 (s, 1H); \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR (125 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ (ppm) 181.6, 163.5, 157.5, 157.1, 152.3, 132.6, 130.3, 129.3, 126.5, 105.2, 105.1, 94.6, and 88.5.\u003c/p\u003e \u003cp\u003e6,8-Dibromoapigenin (\u003cb\u003eF2a\u003c/b\u003e): yellow powder (95% yield). \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR (500 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ (ppm) 8.00 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9.0 Hz, 2H), 6.98 (s, 1H), and 7.95 (m, 2H); \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR (125 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ (ppm) 181.5, 164.3, 161.8, 157.2, 157.1, 152.2, 128.8, 120.8, 116.3, 116.1, 104.9, 94.4, and 88.4.\u003c/p\u003e \u003cp\u003e6,8-Dibromoluteolin (\u003cb\u003eF3a\u003c/b\u003e): yellow powder (97% yield). \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR (500 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ (ppm) 7.52 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10 Hz, 2H), 6.92 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8 Hz, 1H), and and 6.89 (s, 1H); \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR (125 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ (ppm) 181.3, 164.4, 157.4, 157.1, 152.2, 150.3, 145,9, 121.1, 119.4, 116.1, 113.7, 104.7, 102.7, 94.5, and 88.4. HRMS \u003cem\u003em/z\u003c/em\u003e (ESI+): calculated for C\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eBr\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e ([M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e): 442.8766, found 442.8756.\u003c/p\u003e \u003cp\u003e8-Bromobaicalein (\u003cb\u003eF4a\u003c/b\u003e): yellow powder (58% yield). \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR (500 MHz, DMS0-\u003cem\u003ed6\u003c/em\u003e) δ (ppm) 8.12 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9.5 Hz, 2H), 7.61 (m, 3H), and 7.06 (s, 1H); \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR (125 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ (ppm) 182.2, 163.0, 151.6, 146.7, 146.4, 132.3, 130.7, 129.7, 129.4, 126.4, 104.7, and 87.0.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eSynthesis of bromoflavonols\u003c/h2\u003e \u003cp\u003eThe preparation of 6,8-dibromokaempferol (\u003cb\u003eF5a\u003c/b\u003e) and 6,8-dibromomorin (\u003cb\u003eF6a\u003c/b\u003e) were performed by using a previously method.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e By mixing the flavonol (1 mmol) with NaBr (3 mmol) and oxone (3 mmol) in acetone and H\u003csub\u003e2\u003c/sub\u003eO (5:1) for 3 hours. Then, the reaction was stopped by pouring the mixture into H\u003csub\u003e2\u003c/sub\u003eO and treating with Na\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e. The filtration was carried out and washed the residue with H\u003csub\u003e2\u003c/sub\u003eO. Recrystallized the residue with MeOH to get the desired product.\u003c/p\u003e \u003cp\u003eWhile 6,8-dibromoquercetin (\u003cb\u003eF7a\u003c/b\u003e) was obtained as described by a previously method.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e The target compound was synthesized by mixing quercetin (\u003cb\u003eF7\u003c/b\u003e, 1 mmol) with diluted Br\u003csub\u003e2\u003c/sub\u003e (1 mL in 5 mL CH\u003csub\u003e3\u003c/sub\u003eCOOH) at 35\u003csup\u003eo\u003c/sup\u003e C for 3 days. Filtered the precipitate, washed with H\u003csub\u003e2\u003c/sub\u003eO, and recrystallized from MeOH to get the product.\u003c/p\u003e \u003cp\u003e6,8-Dibromokaempferol (\u003cb\u003eF5a\u003c/b\u003e): light yellow powder (34% yield). \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR (500 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ 13.41 (s, 5-OH), 10.29 (s, 3-OH), 9.88 (s, 7-OH), 8.16 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.5 Hz, 2H), and 6.96 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9.0 Hz, 2H); \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR (125 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ (ppm) 175.5, 159.8, 156.9, 156.4, 150.9, 147.8, 136.1, 129.8, 121.6, 115.8, 104.3, 93.7, and 88.0.\u003c/p\u003e \u003cp\u003e6,8-Dibromomorin (\u003cb\u003eF6a\u003c/b\u003e): light yellow powder (53% yield). \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR (500 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ 7.53 (s, 1H), 6.64 (s, 1H), and 6.40 (s, 1H); \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR (125 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ (ppm) 176.2, 160.6, 159.7, 156.8, 156.4, 153.0, 147.7, 136.9, 134.1, 110.7, 104.4, 103.9, 98.6, 98.2, and 86.0.\u003c/p\u003e \u003cp\u003e6,8-Dibromoquercetin (\u003cb\u003eF7a\u003c/b\u003e): light yellow powder (36% yield). \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR (500 MHz, Acetone-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ (ppm) 7.32, (d \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.0 Hz, 1H), 7.19 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0, 2.0 Hz, 1H), and 6.89 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0 Hz, 1H); \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR (125 MHz, Acetone-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ (ppm) 195.5, 160.0, 159.6, 154.6, 147.0, 145.1, 124.8, 121.9, 117.3, 115.2, 108.9, 101.6, 91.7, and 90.9.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eSynthesis of bromoflavanones\u003c/h2\u003e \u003cp\u003eFlavanone (1 mmol) was mixed with acetone and H\u003csub\u003e2\u003c/sub\u003eO (5:1), then NaBr (3 mmol) was added and stirred for 5 minutes. Subsequently, oxone (3 mmol) was added to the mixture solution and stirred at room temperature for 3 h. The mixture was poured into H\u003csub\u003e2\u003c/sub\u003eO, then treated with Na\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e and filtered. The residue was then washed with H\u003csub\u003e2\u003c/sub\u003eO. Recrystallized the residue with MeOH to obtain the target compound.\u003c/p\u003e \u003cp\u003e6,8-Dibromopinocembrin (\u003cb\u003eF8a\u003c/b\u003e): white powder (69% yield). \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR (500 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ 7.54 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.5 Hz, 2H), 7.43 (m, 3H), 5.78 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;12.0, 3.0 Hz, 1H), 3.38 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;17.0, 12.0 Hz, 1H), and 2.99 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;17.0, 3.0 Hz, 1H); \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR (125 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ 196.5, 159.3, 158.5, 157.7, 138.2, 128.8, 126.5, 102.9, 91.0, 90.0, 79.2, 79.1, and 41.2.\u003c/p\u003e \u003cp\u003e6,8-Dibromopinostrobin (\u003cb\u003eF9a\u003c/b\u003e): yellow powder (75% yield). \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR (500 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) δ 7.46 (m, 5H), 5.59 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;12.5, 3.5 Hz, 1H), 3.96 (s, 3H), 3.17 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;17.0, 3.5 Hz, 1H), and 3.04 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;17.0, 3.5 Hz, 1H); \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR (125 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) δ 196.7, 162.5, 159.3, 158.1, 137.5, 129.2, 129.1, 126.0. 106.3, 98.7, 97.0, 79.6, 61.1, and 42.8.\u003c/p\u003e \u003cp\u003e6,8-Dibromoalpinetin (\u003cb\u003eF10a\u003c/b\u003e): white powder (87% yield). \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR (500 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ 7.54 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.0 Hz, 2H), 7.43 (m, 3H), 5.74 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;12.5, 3.5 Hz, 2H), 3.76 (s, 3H), 3.16 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;16.5, 12.5 Hz, 1H), and 2.85 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;17.0, 3.5 Hz, 2H); \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR (125 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ 187.6, 158.8, 157.4, 157.3, 138.5, 128.7, 128.6, 126.3, 109.8, 101.1, 95.7, 78.8, 61.1, and 43.8. HRMS \u003cem\u003em/z\u003c/em\u003e (ESI+): calculated for C\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eBr\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e ([M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e): 426.9181, found 426.9170.\u003c/p\u003e \u003cp\u003e6,8-Dibromonaringenin (\u003cb\u003eF11a\u003c/b\u003e): white powder (65% yield). \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR (500 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ 7.34 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9.0 Hz, 2H), 6.81 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9.0 Hz, 1H), 5.62 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;13.0, 3.5 Hz, 1H), 3.39 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;17.0, 12.0 Hz, 1H), and 2.88 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;17.0, 3.0 Hz, 1H); \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR (DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ 197.0, 159.2, 158.5, 158.0, 157.9, 128.3, 128.3, 115.4, 102.9, 90.8. 89.9, 79.3, and 41.2.\u003c/p\u003e \u003cp\u003e6,8-Dibromohesperetin (\u003cb\u003eF12a\u003c/b\u003e): white powder (98% yield). \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR (500 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ 6.95 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.5 Hz, 2H), 6.89 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.5, 2.5 Hz, 1H), 5.61 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;12.0, 3.0 Hz, 1H), 3.87 (s, 3H), 3.33 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;17.0, 12.0 Hz, 2H), and 2.90 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;17.0, 8.5 Hz, 1H);\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR (125 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ 196.9, 159.3, 158.6, 157.8, 148.2, 146.6, 130.5, 117.9, 114.1, 112.1, 102.9, 90.9, 90.0, 79.2, 55.8, and 41.3.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro α-glucosidase inhibitory activity\u003c/h2\u003e \u003cp\u003eThe determination of \u003cem\u003e α\u003c/em\u003e-glucosidase inhibitory of selected natural flavonoids and their brominated was performed according to the previously described protocol.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e The stock solution of tested compounds and acarbose were dissolved in DMSO and further diluted with phosphate buffer pH 6.9, \u003cem\u003eα\u003c/em\u003e-glucosidase and the substrate \u003cem\u003ep\u003c/em\u003e-NPG were both dissolved in phosphate buffer pH 6.9. About 10 \u003cem\u003e\u0026micro;\u003c/em\u003eL of the tested compound was inserted into a well of 96-wells microplate followed by adding 40 \u003cem\u003e\u0026micro;\u003c/em\u003eL of \u003cem\u003eα\u003c/em\u003e-glucosidase which was then preincubated at 37\u003csup\u003eo\u003c/sup\u003e C for 10 minutes by shaking at 500 rpm. Subsequently, 50 \u003cem\u003e\u0026micro;\u003c/em\u003eL of substrate \u003cem\u003ep\u003c/em\u003e-NPG was added, and the incubation was continued at 37\u003csup\u003eo\u003c/sup\u003e C for 30 minutes by shaking at 500 rpm. Finally, the reaction was terminated by the addition of 100 \u003cem\u003e\u0026micro;\u003c/em\u003eL of 1 M Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e. The absorbance was measured at 405 nm using a microplate reader. Inhibitory activity was calculated by Eq.\u0026nbsp;1.\u003c/p\u003e\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" height=\"47\" width=\"399\"\u003e\u003c/p\u003e\u003cp\u003eWhere A\u003csub\u003e0\u003c/sub\u003e was the absorbance of blank (contained the same volume of the buffer solution instead of the tested compound); A\u003csub\u003et\u003c/sub\u003e was the absorbance of the reaction in the presence of a tested compound, \u003cem\u003eα\u003c/em\u003e-glucosidase, and substrate \u003cem\u003ep-\u003c/em\u003eNPG. The IC\u003csub\u003e50\u003c/sub\u003e values were defined as the concentration of an inhibitor required to inhibit 50% of the \u003cem\u003eα\u003c/em\u003e-glucosidase activity under the assay conditions. The inhibition assay was performed in triplicate and in two independent experiments for all tested compounds.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro α-amylase inhibitory activity\u003c/h2\u003e \u003cp\u003eThe ⍺-amylase assay was performed in 96-well microplates using a final volume of 200 \u003cem\u003e\u0026micro;\u003c/em\u003eL as the previous method with some modification.\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e Unless otherwise stated, experiments were performed with phosphate buffer (0.1 mM) containing 0.02% NaN\u003csub\u003e3\u003c/sub\u003e and adjusted to pH 6.0 with 2.0 M H\u003csub\u003e3\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e. Stock solutions of \u003cem\u003eα\u003c/em\u003e-amylase and CNP-G3 in phosphate buffer were prepared at concentrations of 1 mg/mL and 0.5 mM, respectively. Enzymatic reaction mixtures consisted of test compound (10 \u003cem\u003e\u0026micro;\u003c/em\u003eL), potassium phosphate buffer (140 \u003cem\u003e\u0026micro;\u003c/em\u003eL), an enzyme (20 \u003cem\u003e\u0026micro;\u003c/em\u003eL), then preincubated in phosphate buffer at 37\u003csup\u003eo\u003c/sup\u003e C for 10 minutes by shaking at 500 rpm. Subsequently, the substrate (30 \u003cem\u003e\u0026micro;\u003c/em\u003eL) was incubated at 37\u0026deg; C for 30 minutes. Enzymatic activity was detected by spectrophotometry at 405 nm. Inhibitory activity was calculated by Eq.\u0026nbsp;1. The IC\u003csub\u003e50\u003c/sub\u003e values were defined as the concentration of an inhibitor required to inhibit 50% of the \u003cem\u003eα\u003c/em\u003e-amylase activity under the assay conditions. The IC\u003csub\u003e50\u003c/sub\u003e value was further determined for the tested samples that show the inhibition\u0026thinsp;\u0026ge;\u0026thinsp;70%. For all tested compounds, the inhibition assay was performed in triplicate and two-independent experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eInhibitory kinetic analysis of selected compounds against enzymes\u003c/h2\u003e \u003cp\u003eThe inhibition types of 8-bromobaicalein (\u003cb\u003eF4a\u003c/b\u003e) towards \u003cem\u003eα\u003c/em\u003e-glucosidase and 6,8-dibromoluteolin (\u003cb\u003eF3a\u003c/b\u003e) towards \u003cem\u003eα\u003c/em\u003e-amylase were determined from Lineweaver-Burk plots. Typically, three different concentrations of each compound around the IC\u003csub\u003e50\u003c/sub\u003e value were chosen. The inhibition type was determined using various concentrations of \u003cem\u003ep\u003c/em\u003e-NPG substrate for \u003cem\u003eα\u003c/em\u003e-glucosidase and CNP-G3 for \u003cem\u003eα\u003c/em\u003e-amylase.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eAnti-glycation activity\u003c/h2\u003e \u003cp\u003eIn brief, Bovine Serum Albumin solution (10 mg/mL) was prepared in 0.1 M of phosphate buffer pH 7.4 containing 0.02% NaN\u003csub\u003e3\u003c/sub\u003e. In addition, 14 mM methylglyoxal (MG) was prepared in a phosphate buffer. The test compound and standard inhibitor were prepared in dimethyl sulfoxide (DMSO). About 20 \u003cem\u003e\u0026micro;\u003c/em\u003eL of inhibitor, 80 \u003cem\u003e\u0026micro;\u003c/em\u003eL of phosphate buffer, and 50 \u003cem\u003e\u0026micro;\u003c/em\u003eL of 14 mM MG were mixed in the 96-well and then incubated at 37\u003csup\u003eo\u003c/sup\u003e C for 2 h. After preincubation, 50 \u003cem\u003e\u0026micro;\u003c/em\u003eL of BSA was added to initiate the reaction. The reaction mixture was then incubated at 37\u003csup\u003eo\u003c/sup\u003e C for 1 day. After incubation, each sample was examined for the development of specific fluorescence (excitation 370 nm; emission 450 nm) against a blank (without inhibitor) on a microplate reader and then calculated as Eq.\u0026nbsp;2.\u003c/p\u003e \u003cp\u003e\u003cimg src=\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAXwAAAAsCAYAAABmHgOJAAAAAXNSR0IArs4c6QAAAARnQU1BAACxjwv8YQUAAAAJcEhZcwAADsMAAA7DAcdvqGQAABRQSURBVHhe7d0FsFvF28fxxf64u7u7a3Eo7u6FwV0GBgYYZGAoDoP74O7u7g7Firu7F+h7P0uW9/Q0yb1pb3MD2e/MmSTnJEd3f8+zzz67GW5gByGTyWQy/3mGr7xmMplM5j9OFvxMJpNpE7LgZzKZTJuQY/iZTB369+8fbr/99rD++uuHSSaZpLI2hD/++CM89thj4YEHHggTTjhhGHfcccP3338f/ve//4VNNtkkjDjiiJVvNodXX3013HDDDWHPPfcMI488cmVtJjMo2cPPZGpAwC+99NIwzzzzDCL2IOjjjz9+OOWUU8Lcc88dDcLKK68cvv766zBgwIDKt5rHTDPNFCaYYIJw0UUXVdZkMoOTBT+TqcJff/0Vbrrppvjaq1evytpBefzxx8MMM8wQFllkkfDbb7+F8cYbL2y66aY94mGPMMIIYdVVVw0vvPBCeO655yprM5lBySGdTEvz7LPPhjvuuCOGUH7//few5pprhgUWWKCyddjxxRdfhB133DEcccQRYeaZZ66sHRTi/tNPP4VddtklfPTRR2GppZYK00wzTWVrz3DeeeeFd955Jxx88MHx8xlnnBG++eabMNxww8XQU58+fXLIp43JHn6mpXnyySfDvffeG8YYY4ww2mijNS02/uCDD4axxhqrpth/++234cUXXwy9e/cOn3zySQzlTDbZZJWtPccSSywRPvzww9j34F6NOuqo8d79+OOP4eKLL44GKtO+ZMHPtDRCKmLku+++e9hvv/3i+2agA3SttdaqfBqcp59+Op6b2P1qq60WWx46bHsasXxCL7TDq99mm23CHnvsEbbaaqtowP7888/KNzPtSBb8TEtDtIYffvh/Fp+HNT/88EN45ZVXwqKLLlpZMzhaALPPPnsUURk60003XWVL96MTmGfeVbGeY445wuuvvx5/V7x/zbh3mdYmC34mU+LNN98MY489dsx6qcann34aPegpp5yyKRk577//fgzHaFF0hVlmmSWGdX7++efKmkzmb7LgZzIlPvjggzD55JPX9IhHGmmksO+++4Ytt9wyZscMa/QPvPzyy/G4XYEh0scgcyiTKZIFP5MpQWCFaWoh/37xxRcPc801VxhllFEqawdFlo/somuuuSZ8+eWXMaf/vvvuC1dddVWM/z/yyCPhuuuui6mf119/feyYvvnmm2MoRmaNbZdcckn00nn4jgWhJuGkW2+9NX6uhtaJEJDMpkymSBb8TKaEMM3Qeu5phO60004b4+fHHntsmG+++WI4iDF4++23Y/rkr7/+Gg2D7GhGgcA/+uijYaWVVopG4L333gvPPPNMWH755aPQ33bbbfE7MoNq4Xji/TnjOlMmC36mIbKIdI57JJtISqRQjCkYtAqMI/jqq6/CVFNNFZ544omYt6+D2LgCrQqxd/n8Sy65ZFw/8cQTx30RfSGmW265JWYCyacXTspkGqXLgs/reffdd4eowusEU+i7k++++y4OytHc7W5UyrvuuqtHUtg0w4nBW2+9VVlTHaMpX3rppcqnIYe3WK0zkDjxND/++OP4WYjgiiuuGCxM4HNP5XbzjsWqWw2e+sMPPxxFn3C7h+rN/fffH8uWcqUFYboGn03J8MYbb8Qy/dRTT4Wpp546lj/rxeENlLKfiSaaKIw++uhhzDHHjM+n2WidlHn++edjiOqXX36prPnb4LkO23IaaGvRZcEXczzooIOG6AEaoHLnnXdWPnUPKsepp57a7fvFZ599Fq688soeiYEyrPfcc0/0DGuhQj300EPRMAwNQgTVDIvKe+ihh8b7IKwgxnzWWWfFz+WBT+4RwyArpJkwdvvss08U0VaDFz7OOOPEtM111lknrLfeemHBBRcM8847b9h+++1j9s+uu+4av2M6BAO2vPL8X3vttfjq+9JCib8Rv75rP/LsGQuth2bB2OhP0FFdRP0TmvLsjfBNxve4446L/RaekbmIGOZMa9BlwTcroA6mrszTQZB8jxBgxRVXjMPPuxNNZIXeCMLuQDzVYBuYH+Xwww9v2kAaAqvD7vPPP4+dgERhueWWq2wdHNkjm2++eRz0AxVOc78RY6wDkVFJMeYi1157bYwz8zAJjtCCc9xoo40Gy1zhffI8xaEJQ7PQqUpUGKFWQ7lceOGFYz78pJNOGs9VJ69yZR2B9563Pttss8XfmKDN8zcq1ujeNIDK/DzEn8h7FkbSTj/99E3NqVcmtSqkoib0Kygnyy67bCwn6vvdd98ddYLDos4zYpdffnld5yXTXLok+JpmM844YxxGfuaZZ1bWhigwsgo8fA9ZBoLmvRAO0eJ9EQ7L0IR0hB3sX0hBgRJaIlIKokpB9G3zHefE6+D58yxs4w07N96T7Tq8brzxxpgJoZnKM+Fx8WSl5PmsSZ5yrDXJNbHtw7XB/CS+Y5+8m2JlSGja86KdWwq/OL4OPQJN5N0vvz/66KPjPdKEN9Wt4xBZ5ygjgyftur3XumGg7NP6/fffP1x22WWx0mlK88aEuhyLhy6zowhhcX8ICYEp4/gEyn0nSkRVxSbsZQiPDBLf1aFYD46A63PN5aVWaKkWhFAsu2ys/s0QeXP3NBPPQyvPqzKtD0G5KSIdlJEqZiSpD4yZ8sPo62NQfgm8lg0D5RkZmNaKrbB2pdPaopISFl7HAQccEEM7BBWEZ++9945NODFIr0SMR8irUYAVCINGNP+GlGOOOSaKsKaw5m3yxEEkHIMQEnEFzfn5vjin3yp4vK6jjjoqCvxhhx0W90X4TjzxxHiNcpc1pcVVGQgiaj1BP+GEE2KBdz0777xzjFnKwDDc3ytBdW/cjyLnnHNOnAtG59y2224bPWACTGx5gASSMbBvYsrbc07E3/ecs3CYcArDxvDYh+Wkk06KKX/Wu9fOn/fISJx22mnR+3UvGIHyZFmMAVFP3mUZ3hlDycC4JobPfSEMRKEM0ZcK6FjJSFaDAfaMZKy472lxf08//fR/QgJdwXNvxEB0B8qV8sDJGRZLSt+stq3W4jkOTSuX8yFUo6wKY3IWlKEy5XutHil3qaXhvbLhHjHECS1xnc6Z1qDTmah4dwSNF6bSExsCbjY+QsrzZ8UJscLDQyZCwiGEmGjNP//8UQwIqAIiI4FQquBFD812Qih9LWEdD4HHaZlzzjljihoRsy1hvpDtttsuVhrnqNmrKen8eRw+G/5OmMRVnTNx4u3zknkvvFneDM/XdhBlhsM+sNBCC4ULLrgghqgUcnOVOIZK49X+E7xirRsVgQdFLBgT18HgHHjggfEctKBcjzit9Y6Vro2hME8Lz4sYCAVMMcUUMQSQOqz91rm7BvePkDsOI81bc+1FGA6V2m+qIUvEMTxHFVtzXkvDtVnvumwrYpv9utZqLQEoE56t0ETZM7e/WuczpKSc9SLJQWDUynPcd4YWoE7WZoZTOkPZ7NOnT5h11lkraxqDkyA8yKk4//zzo7PWFTy/ohFI98Rr8f6UP2d6lk49fCLlgfFCeYzixrx44gYP3fqEgpDEKr2Wm4iN4NiMCU+YZy+sRHTK+9QhJiuC92hWRYZIqyN5K8SQENpGOBkGIlPvfF0b48HYpW2asbxeRsJ1E2yL/ZQLNo/WccCQ2A+DmLAvrQr7gmM4ZrEiue/LLLNM6Nu3bxT2JN7l6/dbi+8waASfQBHvMmWxrQbj6DwInD4C5WCDDTaIhrBaJ5xrT+dQC/eJt9evX7/omaaFMFt6IvOkEQgjQ6+V1yoLR2do5/FRjpXNWoa6GsqlepTKKkPPm5damso8lJ+enjI68//UrfkeqAq58cYbhzXWWCOsvvrq8S/UFI5ivDZVciLkvcpfFFKFoihQWgb+Bm6nnXYKO+ywwz8LYS9697APniFDw/P2O95kOlY6BnjbYtniwYSJpyzmnzoUxc2Fp2Sg2MajTuKl0KfCa5/2zyCIT2sF8LChdeIPL8rHLr4HoyC04lx42o6jRcBbPvfcc6PnLE4vRFbsHHYOluL+Ntxww3jujJdzhu3pfNO9Tr/hgVunz6F8PyHeysv1fGuhRSJOq/NYy22xxRaLxkTF1morY18Eo9jCKZPKhdfyUnQaukrxmqvBwBfLl0WZ22KLLRr27iFUwUi30kJ4PZchRQvUs0sx+HrhoeK91kLSwlROlEsOoJa8spr6s7Tg7VtHc6Y1qCv4PHlxPSKX4O1pegvriGV7qJYUStGZyGMllEIfviP2ax8p9t8IxEBnrBASQRfeYGyIMMFU0HiOIKxrr712fIUCqPOYIWGwePdCIkRz6623jkJGeGWsaCGIrXuvwCrcKoCpb3nYOlZlJahcwiyOzfAp9O6J80hGAe4Rz8tf4IlRq5xS1BzXfeJ5i8M7rr4BhsGQenFZ+7SvZIzsR7M9hZXcS/dUZfU7Ysyj96yct3PUEhL6qpZppFVhX/WyJ7SO3HuC4lwZMPfduZbF2TE9fy2sYsdeGeelP4PgMtxp8UciDDpj2FVSLF2Zqme4hhVF8UtG+t+E81enjj/++LDKKqvEOiOOXy1+zwikJAb/8qV+C616ZsJAWt9a3eoKZ4jDYb36z0lUxjOtQd1/vPJgFWReWYrZ8nzLHmgR64UvvCo8vEH7IR7WNeqNEHbGhdgTWMKq8PlMXOwzeefEmyjrTC3id45P8KHgElMCQzCdk2v0Pfu0L9/3ms6XSLv25GFXCz/4jfvkFb6f7oF9pf25HyqRVojjwnm4Dp9dk8W+rPPeb9LkWd7bt+OkZ0P0Ugzc91XeFVZYYbD4fULLR2vHPOm8/TI8eftLBoMBch1aZ0UcS8cy48i704rpTnTmciJ0ZBfhWbr21BJL5bM7OPvss2PLlhjWQnlw3YwuowiGWCix1j1vFuqMkbhEl+cOxnGvvfaKIpw6VSUIeMYW5U+dKD9fuMfKXLrfykQqt/ptPAthm1TuwWnxHYYg0zrU9fBVIg+3WJkIEOGxvtqSxNP3CIlX69K2RuFJ67S9+uqro6gQfB6EQprOwzodoAq4sE8Z301iD79L3qT1zlFhdb7pmn2neL6+n8Qe6XqLi98UC33xHhT35xj2lSoNnIft6Zq89zvYp/UJv7fd99KzScZDFoywm/X1YrI8bSEPLQWGqAzhtv8E4agmBio74ZBz3d1iX490rxjodA+aieclBVF2Fjwr4TnC+m9B+VA34HqqPV+4tuL9LpZbxs5YjmK5h1ZkFvvWo67gtwKaiEIoPE4LUdGcLBY6BVHIRMaB8EY74/6oaAbDdBYiYTiFuIYGYivrSFpoO0EEedI6smVuCR9qWTKkmUyr0vKCT8z91Zy/txOPJ+hlb0ITVbaC7JR2xn1Zeuml471KTfnO4OEVjWejEPxi66ld0KrUB8IhkXWk/4eHnDzmTKYVaXnBz2RaEfMY8fKFHHXop5h1JtPK5BKayZTQqqzWr1FEvF7Kq1anSdx69epV2dLz6FhljMot4XbBdVdbhjZ8OTQM6+Pbd/Faa2VEZsHPZEoYQKS/qBY6qqXuSldN/UcpFbiZyJCqhhRaYbae6MxuFWRKuT/FRWusWejQL2eWDSuMLZF8ITXbdUpDr5UplgU/kymhA1q6YS2M1ZCS2cjI1O5ECu6FF144yPiYIkZzywKTWZNpPsTeYNUiw9LgGOBp/2mAWxrZ7DzKZMHPZEpoHqfBhGVULAPhjHOQLeZzdyLFtTOMATFtRq3OcoPSZGq1Y2d6VyGGKfzBQ/ZaDLmUP6eQScJYoPR7i7+kTOuT2EueSPso70/Ipfh755BI2+zTkr7T1YGrJl6sRRb8TKaEsRPGKFT7gxkVz2hjo1MJv8/VEEc3ktvkbQY1CQP5hzYjVYVcZPiYytpiRlV9Bjw1s76ab8hkgyouA2CQl/+WsI7YG2Ao973aFBcwoM6cNsJN7YoQRxLKtKQQC+EkykajM9hCc42iz0boxO9N52L6FkaEl+0zzH9VzatnFIRc0u+dqwGGyWgk7NMkk0I1KBqFWrhGv4N04TJZ8DMtTy1RHZaYwsMMoUOCUakGwJleWvqmEIvpNRgS/x3hj0J0+qr4QkO+K8OH+Bs/QZCEbISViD/hIODmUyL8BiDWyvfn3Tu+qU2KtFsGUbUYfvrHLoM4YZQ5/LFPo9ifznplM3n0MrW6gilW4PfgOBB/Ql304hkkdHUuIgZHqwL2Z79lsuBnWhoVi4BZTInRWfZMd8GDEyvnpTeKAVk8M2MizDdDtO1Ly8CUGsSXt29MCS9c6MUUHHL49QswEnL6N9tss2gYDChkLLQUjH7W8qg2ohyMiFGujoV077QwmnXv/i0MzUhgQs+o8L6TR99V6oVm6vUd1esDKIaSnJfWYjWy4GdaGhVLaENz1sRz5gBqBjJ1DPhL3lgjEFZhGMKtY9V0C+arV2GFYQiykI/MHvMEOZZrJPYm4mMgklfnN7x7Iq8zzlQGRIH4l2FohH3WXXfdaEiIvP+h5u0dcsghsU9Cumbmb9zrISF1hhL6at53Z4akmuedSPMyNUoxLbieYciCn2lpzLZ45JFHxpg5AW7W3Oq8cLM/Mji8p0Yw9bKR3yYjI7xmcCXmvHxirMLvtttu0bMXOtIS0KHn+njyph5O8VdiLebP+zeSXAqof5krz3vDgAj5mJnSn+OAuJs+wzxHZigVVkp/7NPO+DN4mEgO1dInecli6/D80/si6c910n66ihli4b87wOMXw68VhumMYmy/2nkqw/90GHc0mTOZTA369+8/8OSTTx7Y4T1X1nSdDhGuvBs4sMPbHjhgwIDKp8bo8OYH2Vc1+vXrN7DDMA7sMDKVNe0LWau1dAhi/E6Hd/7Pur59+8bXDpGP21Dc3iHEcZv3id69ew+y3at1ifT9tM/ieziP9HuLfSTStuL+yr8vko5VXlwDvE+/rTs9ciaTybQDKSZfLxzyXyCHdDKZTKZNyIKfyWQybUIO6WQymUxbEML/AX0RLi4MAenIAAAAAElFTkSuQmCC\" height=\"44\" width=\"380\"\u003e\u003c/p\u003e\u003cp\u003eAfterward, the IC\u003csub\u003e50\u003c/sub\u003e value was further investigated for the tested compounds that show inhibition\u0026thinsp;\u0026ge;\u0026thinsp;80%. For all tested compounds, the inhibition assay was performed in triplicate.\u003c/p\u003e \u003cp\u003e \u003cem\u003eStatistical Analysis.\u003c/em\u003e All the experiments were carried out in triplicate and in two independent experiments. The inhibition percentage and or IC\u003csub\u003e50\u003c/sub\u003e values were calculated by using Microsoft Excel version 16.64 and a GraphPad Prism version 9. The data are expressed as means of two independent experiments\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviations. \u003csup\u003ec\u003c/sup\u003eLog P values were obtained from ChemDraw Professional 16.0.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eWe appreciate Chulalongkorn University–Graduate Programme Scholarship for ASEAN and NON-ASEAN Countries Academic Year 2019 for supporting Ms. Rita Hairani to study at Chulalongkorn University. Ms. R. Hairani is also grateful to Graduate School Thesis Grant, Chulalongkorn University for financial support.\u003c/p\u003e\n\u003cp\u003eAuthor\u0026nbsp;contributions statement\u003c/p\u003e\n\u003cp\u003eR.H. performed all experiments including preparation of all compounds, in vitro\u0026nbsp;\u003cem\u003e⍺\u003c/em\u003e-glucosidase and\u0026nbsp;\u003cem\u003e⍺\u003c/em\u003e-amylase inhibitory activity assay, as well as anti-glycation activity. W.C. designed the synthesis of bromoflavonoids, supervised the study, provided critical discussion, and prepared the manuscript to be published. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003eCompeting interest\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003eData Availability\u003c/p\u003e\n\u003cp\u003eThe data supporting the findings of this study are available within the paper and its Supplementary Information files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRammohan, A., Bhaskar, B. V., Venkateswarlu, N., Gu, W. \u0026amp; Zyryanov, G. V. Design, synthesis, docking and biological evaluation of chalcones as promising antidiabetic agents. \u003cem\u003eBioorg. 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Synthesis of 4-methoxybenzoylhydrazones and evaluation of their antiglycation activity. \u003cem\u003eMolecules\u003c/em\u003e \u003cb\u003e19\u003c/b\u003e, 1286\u0026ndash;1301. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/molecules19011286\u003c/span\u003e\u003cspan address=\"10.3390/molecules19011286\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2014).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"8-bromobaicalein, 6,8-dibromoluteolin, 6,8-dibromochrysin, α-glucosidase, α-amylase, anti-glycation","lastPublishedDoi":"10.21203/rs.3.rs-6452882/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6452882/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTwelve selected natural flavonoids were investigated for their antidiabetic effects on \u003cem\u003eα\u003c/em\u003e-glucosidase and \u003cem\u003eα\u003c/em\u003e-amylase, as well as anti-glycation activity. A series of brominated analogues from these selected flavonoids were synthesized. Two new semisynthetic compounds including 6,8-dibromoluteolin (\u003cb\u003eF3a\u003c/b\u003e) and 6,8-dibromoalpinetin (\u003cb\u003eF10a\u003c/b\u003e) have been synthesized. 8-Bromobaicalein (\u003cb\u003eF4a\u003c/b\u003e, IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 \u0026micro;M) and 6,8-dibromoluteolin (\u003cb\u003eF3a\u003c/b\u003e, IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 \u0026micro;M) were found as mixed-type potent agents on \u003cem\u003eα\u003c/em\u003e-glucosidase and \u003cem\u003eα\u003c/em\u003e-amylase inhibitory activities, respectively. In addition, 6,8-dibromochrysin (\u003cb\u003eF1a\u003c/b\u003e, IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;50.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98 \u0026micro;M) was found to be the most potent compound for the inhibition of Bovine Serum Albumin - glycation (BSA-glycation) mediated methylglyoxal.\u003c/p\u003e","manuscriptTitle":"Synthesis of promising brominated flavonoids as antidiabetic and anti-glycation agents","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-30 13:49:37","doi":"10.21203/rs.3.rs-6452882/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-23T03:58:55+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-17T07:21:37+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-12T11:33:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"179498910166156087278459646120681113869","date":"2025-05-02T05:39:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"236044394282139306157209913675140970106","date":"2025-04-29T05:11:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"260485213848408515171458624970193243872","date":"2025-04-29T04:18:02+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-28T16:59:52+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-28T12:40:02+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-04-28T10:31:40+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-28T10:29:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-04-15T08:48:02+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5f095f70-9433-4348-b6d3-a0975f1d7964","owner":[],"postedDate":"April 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":47830690,"name":"Biological sciences/Drug discovery"},{"id":47830691,"name":"Health sciences/Diseases"},{"id":47830692,"name":"Physical sciences/Chemistry"}],"tags":[],"updatedAt":"2025-07-21T16:08:56+00:00","versionOfRecord":{"articleIdentity":"rs-6452882","link":"https://doi.org/10.1038/s41598-025-09040-9","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-07-15 15:57:06","publishedOnDateReadable":"July 15th, 2025"},"versionCreatedAt":"2025-04-30 13:49:37","video":"","vorDoi":"10.1038/s41598-025-09040-9","vorDoiUrl":"https://doi.org/10.1038/s41598-025-09040-9","workflowStages":[]},"version":"v1","identity":"rs-6452882","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6452882","identity":"rs-6452882","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}